Highly conserved tuf genes and their use to generate probes and primers for detection of coagulase-negative Staphylococcus

ABSTRACT

Provided herein are compositions and methods for the detection of Streptococcus agalacticae.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/867,986,filed Apr. 22, 2013, which is a continuation of application Ser. No.13/176,626, filed Jul. 5, 2011, now U.S. Pat. No. 8,426,137, which is acontinuation of application Ser. No. 11/522,253, filed Sep. 14, 2006,which is a continuation-in-part of application Ser. No. 11/236,785,filed Sep. 27, 2005, now U.S. Pat. No. 8,114,601, which is acontinuation of Ser. No. 10/089,177, filed Mar. 27, 2002, which is theU.S. national phase under 35 U.S.C. § 371 of prior PCT InternationalApplication No. PCT/CA2000/001150, filed Sep. 28, 2000, which claims thebenefit of Canada Application No. 2307010 filed May 19, 2000, and CanadaApplication No. 2283458, filed Sep. 28, 1999, the disclosures of whichare hereby expressly incorporated by reference in their entireties.

SEQUENCE LISTING

The present application is being filed along with a sequence listing inelectronic format. The sequence listing is provided as a file entitledGENOM_048P1C3_Substitute_sequence_listing.txt, created Apr. 18, 2017which is 2.30 MB in size. The information in the electronic format ofthe sequence listing is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Classical Methods for the Identification of Microorganisms

Microorganisms are classically identified by their ability to utilizedifferent substrates as a source of carbon and nitrogen through the useof biochemical tests such as the API20E™ system (bioMérieux). Forsusceptibility testing, clinical microbiology laboratories use methodsincluding disk diffusion, agar dilution and broth microdilution.Although identifications based on biochemical tests and antibacterialsusceptibility tests are cost-effective, generally two days are requiredto obtain preliminary results due to the necessity of two successiveovernight incubations to identify the bacteria from clinical specimensas well as to determine their susceptibility to antimicrobial agents.There are some commercially available automated systems (i.e. theMicroScan™ system from Dade Behring and the Vitek™ system frombioMérieux) which use sophisticated and expensive apparatus for fastermicrobial identification and susceptibility testing (Stager and Davis,1992, Clin. Microbiol. Rev. 5:302-327). These systems require shorterincubation periods, thereby allowing most bacterial identifications andsusceptibility testing to be performed in less than 6 hours.Nevertheless, these faster systems always require the primary isolationof the bacteria or fungi as a pure culture, a process which takes atleast 18 hours for a pure culture or 2 days for a mixed culture. So, theshortest time from sample reception to identification of the pathogen isaround 24 hours. Moreover, fungi other than yeasts are often difficultor very slow to grow from clinical specimens. Identification must relyon labor-intensive techniques such as direct microscopic examination ofthe specimens and by direct and/or indirect immunological assays.Cultivation of most parasites is impractical in the clinical laboratory.Hence, microscopic examination of the specimen, a few immunologicaltests and clinical symptoms are often the only methods used for anidentification that frequently remains presumptive.

The fastest bacterial identification system, the autoSCAN-Walk-Away™system (Dade Behring) identifies both gram-negative and gram-positivebacterial species from standardized inoculum in as little as 2 hours andgives susceptibility patterns to most antibiotics in 5 to 6 hours.However, this system has a particularly high percentage (i.e. 3.3 to40.5%) of non-conclusive identifications with bacterial species otherthan Enterobacteriaceae (Croizé J., 1995, Lett. Infectiol. 10:109-113;York et al., 1992, J. Clin. Microbiol. 30:2903-2910). ForEnterobacteriaceae, the percentage of non-conclusive identifications was2.7 to 11.4%. The list of microorganisms identified by commercialsystems based on classical identification methods is given in Table 15.

A wide variety of bacteria and fungi are routinely isolated andidentified from clinical specimens in microbiology laboratories. Tables1 and 2 give the incidence for the most commonly isolated bacterial andfungal pathogens from various types of clinical specimens. Thesepathogens are the main organisms associated with nosocomial andcommunity-acquired human infections and are therefore considered themost clinically important.

Clinical Specimens Tested in Clinical Microbiology Laboratories

Most clinical specimens received in clinical microbiology laboratoriesare urine and blood samples. At the microbiology laboratory of theCentre Hospitalier de l'Université Laval (CHUL), urine and blood accountfor approximately 55% and 30% of the specimens received, respectively(Table 3). The remaining 15% of clinical specimens comprise variousbiological fluids including sputum, pus, cerebrospinal fluid, synovialfluid, and others (Table 3). Infections of the urinary tract, therespiratory tract and the bloodstream are usually of bacterial etiologyand require antimicrobial therapy. In fact, all clinical samplesreceived in the clinical microbiology laboratory are tested routinelyfor the identification of bacteria and antibiotic susceptibility.

Conventional Pathogen Identification from Clinical Specimens

Urine Specimens

The search for pathogens in urine specimens is so preponderant in theroutine microbiology laboratory that a myriad of tests have beendeveloped. However, the gold standard remains the classicalsemi-quantitative plate culture method in which 1 μL of urine isstreaked on agar plates and incubated for 18-24 hours. Colonies are thencounted to determine the total number of colony forming units (CFU) perliter of urine. A bacterial urinary tract infection (UTI) is normallyassociated with a bacterial count of 10⁷ CFU/L or more in urine.However, infections with less than 10⁷ CFU/L in urine are possible,particularly in patients with a high incidence of diseases or thosecatheterized (Stark and Maki, 1984, N. Engl. J. Med. 311:560-564).Importantly, approximately 80% of urine specimens tested in clinicalmicrobiology laboratories are considered negative (i.e. bacterial countof less than 10⁷ CFU/L; Table 3). Urine specimens found positive byculture are further characterized using standard biochemical tests toidentify the bacterial pathogen and are also tested for susceptibilityto antibiotics. The biochemical and susceptibility testing normallyrequire 18-24 hours of incubation.

Accurate and rapid urine screening methods for bacterial pathogens wouldallow a faster identification of negative specimens and a more efficienttreatment and care management of patients. Several rapid identificationmethods (Uriscreen™, UTIscreen™, Flash Track™ DNA probes and others)have been compared to slower standard biochemical methods, which arebased on culture of the bacterial pathogens. Although much faster, theserapid tests showed low sensitivities and poor specificities as well as ahigh number of false negative and false positive results (Koening etal., 1992, J. Clin. Microbiol. 30:342-345; Pezzlo et al., 1992, J. Clin.Microbiol. 30:640-684).

Blood Specimens

The Blood Specimens Received In The Microbiology Laboratory Are AlwaysSubmitted For Culture. Blood Culture Systems May Be Manual,Semi-Automated Or Completely Automated. The BACTEC™ System (From BectonDickinson) And The Bactalert™ System (From Organon Teknika Corporation)Are The Two Most Widely Used Automated Blood Culture Systems. TheseSystems Incubate Blood Culture Bottles Under Optimal Conditions ForGrowth Of Most Bacteria. Bacterial Growth Is Monitored Continuously ToDetect Early Positives By Using Highly Sensitive Bacterial GrowthDetectors. Once Growth Is Detected, A Gram Stain Is Performed DirectlyFrom The Blood Culture And Then Used To Inoculate Nutrient Agar Plates.Subsequently, Bacterial Identification And Susceptibility Testing AreCarried Out From Isolated Bacterial Colonies With Automated Systems AsDescribed Previously. Blood Culture Bottles Are Normally Reported AsNegative If No Growth Is Detected After An Incubation Of 6 To 7 Days.Normally, The Vast Majority Of Blood Cultures Are Reported Negative. ForExample, The Percentage Of Negative Blood Cultures At The MicrobiologyLaboratory Of The CHUL For The Period February 1994-January 1995 Was93.1% (Table 3).

Other Clinical Samples

Upon receipt by the clinical microbiology laboratory, all body fluidsother than blood and urine that are from normally sterile sites (i.e.cerebrospinal, synovial, pleural, pericardial and others) are processedfor direct microscopic examination and subsequent culture. Again, mostclinical samples are negative for culture (Table 3). In all thesenormally sterile sites, tests for the universal detection of algae,archaea, bacteria, fungi and parasites would be very useful.

Regarding clinical specimens which are not from sterile sites such assputum or stool specimens, the laboratory diagnosis by culture is moreproblematic because of the contamination by the normal flora. Thebacterial or fungal pathogens potentially associated with the infectionare grown and separated from the colonizing microbes using selectivemethods and then identified as described previously. Of course, theDNA-based universal detection of bacteria would not be useful for thediagnosis of bacterial infections at these non-sterile sites. On theother hand, DNA-based assays for species or genus or family or groupdetection and identification as well as for the detection ofantimicrobial agents resistance genes from these specimens would be veryuseful and would offer several advantages over classical identificationand susceptibility testing methods.

DNA-Based Assays with any Specimen

There is an obvious need for rapid and accurate diagnostic tests for thedetection and identification of algae, archaea, bacteria, fungi andparasites directly from clinical specimens. DNA-based technologies arerapid and accurate and offer a great potential to improve the diagnosisof infectious diseases (Persing et al., 1993, Diagnostic MolecularMicrobiology: Principles and Applications, American Society forMicrobiology, Washington, D.C.; Bergeron and Ouellette, 1995, Infection23:69-72; Bergeron and Ouellette, 1998, J Clin Microbiol. 36:2169-72).The DNA probes and amplification primers which are objects of thepresent invention are applicable for the detection and identification ofalgae, archaea, bacteria, fungi, and parasites directly from anyclinical specimen such as blood, urine, sputum, cerebrospinal fluid,pus, genital and gastro-intestinal tracts, skin or any other type ofspecimens (Table 3). These assays are also applicable to detection frommicrobial cultures (e.g. blood cultures, bacterial or fungal colonies onnutrient agar, or liquid cell cultures in nutrient broth). The DNA-basedtests proposed in this invention are superior in terms of both rapidityand accuracy to standard biochemical methods currently used for routinediagnosis from any clinical specimens in microbiology laboratories.Since these tests can be performed in one hour or less, they provide theclinician with new diagnostic tools which should contribute to a bettermanagement of patients with infectious diseases. Specimens from sourcesother than humans (e.g. other primates, birds, plants, mammals, farmanimals, livestock, food products, environment such as water or soil,and others) may also be tested with these assays.

A High Percentage of Culture-Negative Specimens

Among all the clinical specimens received for routine diagnosis,approximately 80% of urine specimens and even more (around 95%) forother types of normally sterile clinical specimens are negative for thepresence of bacterial pathogens (Table 3). It would also be desirable,in addition to identify bacteria at the species or genus or family orgroup level in a given specimen, to screen out the high proportion ofnegative clinical specimens with a DNA-based test detecting the presenceof any bacterium (i.e. universal bacterial detection). As disclosed inthe present invention, such a screening test may be based on DNAamplification by PCR of a highly conserved genetic target found in allbacteria. Specimens negative for bacteria would not be amplified by thisassay. On the other hand, those that are positive for any bacteriumwould give a positive amplification signal. Similarly, highly conservedgenes of fungi and parasites could serve not only to identify particularspecies or genus or family or group but also to detect the presence ofany fungi or parasite in the specimen.

Towards the Development of Rapid DNA-Based Diagnostic Tests

A rapid diagnostic test should have a significant impact on themanagement of infections. DNA probe and DNA amplification technologiesoffer several advantages over conventional methods for theidentification of pathogens and antimicrobial agents resistance genesfrom clinical samples (Persing et al., 1993, Diagnostic MolecularMicrobiology: Principles and Applications, American Society forMicrobiology, Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-basedDiagnostics in Infectious Disease, Blackwell Scientific Publications,Boston, Mass.). There is no need for culture of the pathogens, hence theorganisms can be detected directly from clinical samples, therebyreducing the time associated with the isolation and identification ofpathogens. Furthermore, DNA-based assays are more accurate for microbialidentification than currently used phenotypic identification systemswhich are based on biochemical tests and/or microscopic examination.Commercially available DNA-based technologies are currently used inclinical microbiology laboratories, mainly for the detection andidentification of fastidious bacterial pathogens such as Mycobacteriumtuberculosis, Chlamydia trachomatis, Neisseria gonorrhoeae as well asfor the detection of a variety of viruses (Tang Y. and Persing D. H.,Molecular detection and identification of microorganisms, In: P. Murrayet al., 1999, Manual of Clinical Microbiology, ASM press, 7^(th)edition, Washington D.C.). There are also other commercially availableDNA-based assays which are used for culture confirmation assays.

Others have developed DNA-based tests for the detection andidentification of bacterial pathogens which are objects of the presentinvention, for example: Staphylococcus sp. (U.S. Pat. No. 5,437,978),Neisseria sp. (U.S. Pat. No. 5,162,199 and European patent serial no.0,337,896,131) and Listeria monocytogenes (U.S. Pat. Nos. 5,389,513 and5,089,386). However, the diagnostic tests described in these patents arebased either on rRNA genes or on genetic targets different from thosedescribed in the present invention. To our knowledge there are only fourpatents published by others mentioning the use of any of the four highlyconserved gene targets described in the present invention for diagnosticpurposes (PCT international publication number WO92/03455 andWO00/14274, European patent publication number 0 133 671 B1, andEuropean patent publication number 0 133 288 A2). WO92/03455 is focusedon the inhibition of Candida species for therapeutic purposes. Itdescribes antisense oligonucleotide probes hybridizing to Candidamessenger RNA. Two of the numerous mRNA proposed as targets are codingfor translation elongation factor 1 (tef1) and the beta subunit ofATPase. DNA amplification or hybrization are not under the scope oftheir invention and although diagnostic use is briefly mentioned in thebody of the application, no specific claim is made regardingdiagnostics. WO00/14274 describes the use of bacterial recA gene foridentification and speciation of bacteria of the Burkholderia cepaciacomplex. Specific claims are made on a method for obtaining nucleotidesequence information for the recA gene from the target bacteria and afollowing comparison with a standard library of nucleotide sequenceinformation (claim 1), and on the use of PCR for amplification of therecA gene in a sample of interest (claims 4 to 7, and 13). However, theuse of a discriminatory restriction enzyme in a RFLP procedure isessential to fulfill the speciation and WO00/14274 did not mention thatmultiple recA probes could be used simultaneously. Patent EP 0 133 288A2 describes and claims the use of bacterial tuf (and fus) sequence fordiagnostics based on hybridization of a tuf (or fus) probe withbacterial DNA. DNA amplification is not under the scope of EP 0 133 288A2. Nowhere it is mentioned that multiple tuf (or fus) probes could beused simultaneously. No mention is made regarding speciation using tuf(or fus) DNA nucleic acids and/or sequences. The sensitivities of thetuf hybrizations reported are 1×10⁶ bacteria or 1-100 ng of DNA. This ismuch less sensitive than what is achieved by our assays using nucleicacid amplification technologies.

Although there are phenotypic identification methods which have beenused for more than 125 years in clinical microbiology laboratories,these methods do not provide information fast enough to be useful in theinitial management of patients. There is a need to increase the speed ofthe diagnosis of commonly encountered bacterial, fungal and parasiticalinfections. Besides being much faster, DNA-based diagnostic tests aremore accurate than standard biochemical tests presently used fordiagnosis because the microbial genotype (e.g. DNA level) is more stablethan the phenotype (e.g. physiologic level).

Bacteria, fungi and parasites encompass numerous well-known microbialpathogens. Other microorganisms could also be pathogens or associatedwith human diseases. For example, achlorophylious algae of thePrototheca genus can infect humans. Archae, especially methanogens, arepresent in the gut flora of humans (Reeve, J. H., 1999, J. Bacteriol.181:3613-3617). However, methanogens have been associated to pathologicmanifestations in the colon, vagina, and mouth (Belay et al., 1988,Appl. Enviro. Microbiol. 54:600-603; Belay et al., 1990, J. Clin.Microbiol. 28:1666-1668; Weaver et al., 1986, Gut 27:698-704).

In addition to the identification of the infectious agent, it is oftendesirable to identify harmful toxins and/or to monitor the sensitivityof the microorganism to antimicrobial agents. As revealed in thisinvention, genetic identification of the microorganism could beperformed simultaneously with toxin and antimicrobial agents resistancegenes. Alternatively, assays to identify toxin and/or antimicrobialresistance genes can be performed separately and independently fromassays for identification of infectious agents.

Knowledge of the genomic sequences of algal, archaeal, bacterial, fungaland parasitical species continuously increases as testified by thenumber of sequences available from public databases such as GenBank.From the sequences readily available from those public databases, thereis no indication therefrom as to their potential for diagnosticpurposes. For determining good candidates for diagnostic purposes, onecould select sequences for DNA-based assays for (i) the species-specificdetection and identification of commonly encountered bacterial, fungaland parasitical pathogens, (ii) the genus-specific detection andidentification of commonly encountered bacterial, fungal or parasiticalpathogens, (iii) the family-specific detection and identification ofcommonly encountered bacterial, fungal or parasitical pathogens, (iv)the group-specific detection and identification of commonly encounteredbacterial, fungal or parasitical pathogens, (v) the universal detectionof algal, archaeal, bacterial, fungal or parasitical pathogens, and/or(vi) the specific detection and identification of antimicrobial agentsresistance genes, and/or (vii) the specific detection and identificationof bacterial toxin genes. All of the above types of DNA-based assays maybe performed directly from any type of clinical specimens or from amicrobial culture.

In our assigned U.S. Pat. No. 6,001,564 and our WO98/20157 patentpublication, we described DNA sequences suitable for (i) thespecies-specific detection and identification of clinically importantbacterial pathogens, (ii) the universal detection of bacteria, and (iii)the detection of antimicrobial agents resistance genes.

The WO98/20157 patent publication describes proprietary tuf DNAsequences as well as tuf sequences selected from public databases (inboth cases, fragments of at least 100 base pairs), as well asoligonucleotide probes and amplification primers derived from thesesequences. All the nucleic acid sequences described in that patentpublication can enter in the composition of diagnostic kits or productsand methods capable of a) detecting the presence of bacteria and fungib) detecting specifically at the species, genus, family or group levels,the presence of bacteria and fungi and antimicrobial agents resistancegenes associated with these pathogens. However, these methods and kitsneed to be improved, since the ideal kit and method should be capable ofdiagnosing close to 100% of microbial pathogens and associatedantimicrobial agents resistance genes and toxins genes. For example,infections caused by Enterococcus faecium have become a clinical problembecause of its resistance to many antibiotics. Both the detection ofthese bacteria and the evaluation of their resistance profiles aredesirable. Besides that, novel DNA sequences (probes and primers)capable of recognizing the same and other microbial pathogens or thesame and additional antimicrobial agents resistance genes are alsodesirable to aim at detecting more target genes and complement ourearlier patent applications.

The present invention improves the assigned application by disclosingnew proprietary tuf nucleic acids and/or sequences as well as describingnew ways to obtain tuf nucleic acids and/or sequences. In addition wedisclose new proprietary atpD and recA nucleic acids and/or sequences.In addition, new uses of tuf, atpD and recA DNA nucleic acids and/orsequences selected from public databases (Table 11) are disclosed.

Highly Conserved Genes for Identification and Diagnostics

Highly conserved genes are useful for identification of microorganisms.For bacteria, the most studied genes for identification ofmicroorganisms are the universally conserved ribosomal RNA genes (rRNA).Among those, the principal targets used for identification purposes arethe small subunit (SSU) ribosomal 16S rRNA genes (in prokaryotes) and18S rRNA genes (in eukaryotes) (Relman and Persing, Genotyping Methodsfor Microbial Identification, In: D. H. Persing, 1996, PCR Protocols forEmerging Infectious Diseases, ASM Press, Washington D.C.). The rRNAgenes are also the most commonly used targets for universal detection ofbacteria (Chen et al., 1988, FEMS Microbiol. Lett. 57:19-24; McCabe etal., 1999, Mol. Genet. Metabol. 66:205-211) and fungi (Van Burik et al.,1998, J. Clin. Microbiol. 36:1169-1175).

However, it may be difficult to discriminate between closely relatedspecies when using primers derived from the 16S rRNA. In some instances,16S rRNA sequence identity may not be sufficient to guarantee speciesidentity (Fox et al., 1992, Int. J. Syst. Bacteriol. 42:166-170) and ithas been shown that inter-operon sequence variation as well as strain tostrain variation could undermine the application of 16S rRNA foridentification purposes (Clayton et al., 1995, Int. J. Syst. Bacteriol.45:595-599). The heat shock proteins (HSP) are another family of veryconserved proteins. These ubiquitous proteins in bacteria and eukaryotesare expressed in answer to external stress agents. One of the mostdescribed of these HSP is HSP 60. This protein is very conserved at theamino acid level, hence it has been useful for phylogenetic studies.Similar to 16S rRNA, it would be difficult to discriminate betweenspecies using the HSP 60 nucleotide sequences as a diagnostic tool.However, Goh et al. identified a highly conserved region flanking avariable region in HSP 60, which led to the design of universal primersamplifying this variable region (Goh et al., U.S. Pat. No. 5,708,160).The sequence variations in the resulting amplicons were found useful forthe design of species-specific assays.

SUMMARY OF THE INVENTION

Disclosed herein are compositions and methods for the detection andquantitation of antibiotic resistant organisms from a sample.

Some embodiments relate to compositions for the detection of avancomycin resistant pathogen in a sample using a nucleic acidamplification assay. The composition can include at least one primerpair, comprising a forward and a reverse oligonucleotide, wherein theforward and reverse oligonucleotides each includes a binding region thatis complementary to primer binding sites present on opposite strands ofthe pathogen's DNA, wherein the primer pair is adapted to amplify a vanAor vanB gene of the pathogen's DNA between and including said primerbinding sites to produce a detectable amplification product. The bindingregions of the oligonucleotides can correspond to, or be fullycomplementary to, at least 10, 11, 12, 13, 14, 15, 16, 17, 18., 19, or20 consecutive nucleotides of at least two of the following sequences:SEQ ID NO: 1090, SEQ ID NO: 1091, SEQ ID NO: 1095, SEQ ID NO: 2298 andSEQ ID NO: 1096.

In some embodiments, the composition includes a primer pair wherein theprimers comprise, consist essentially of, or consist of at least 10consecutive nucleotides of SEQ ID NO: 1095 and SEQ ID NO: 1096.Preferred compositions include a primer pair wherein the primerscomprises, consists essentially of, or consist of the sequences of SEQID NO: 1095 and SEQ ID NO: 1096.

Preferably, the composition comprises at least four primers, wherein theeach of the four primers primers comprises, consists essentially of, orconsists of at least 10 consecutive nucleotides of SEQ ID NOs: 1090,1091, 1096 and 2298, or the complements thereof. In other preferredembodiments, the four primers comprise, consist essentially of, orconsist of the SEQ ID NO: 1090, 1091, 1096, and 2298.

In some embodiments, the composition can also include at least oneinternal hybridization probe, wherein the internal hybridization probecan hybridize under stringent condition to the vanA or vanBamplification products produced by the compositions described above.Preferably, the internal hybridization probe is a molecular beacon. Inmore preferred embodiments, the molecular beacon can include thesequence of SEQ ID NO: 2299 or SEQ ID NO: 2300.

In further embodiments, the compositions can also include at least oneinternal control DNA, which can be amplified to produce an internalcontrol amplicon under the same conditions and using the sameoligonucleotides of the compositions described above. In furtherembodiments, the compositions can include an internal control probe thatcan hybridize under stringent conditions to the internal controlamplicon. In some embodiments, the internal control DNA comprises thesequence of SEQ ID NO: 2302. Preferably, the internal control probe is amolecular beacon. In some embodiments, the internal control probeincludes at least 10 consecutive nucleotides of the sequence of SEQ IDNO: 2301.

Also provided herein are kits that include the compositions describedherein.

Methods to detect the presence of vancomycin-resistant organisms in asample are also provided. In some embodiments, the method can includethe step of annealing the nucleic acids of the sample with at least oneprobe and/or primer, wherein each of the primers and/or probes includenucleic acid sequences that correspond to, or are fully complementaryto, at least 10 consecutive nucleotides of at least two of the followingsequences: SEQ ID NO: 1090, SEQ ID NO: 1091, SEQ ID NO: 1095 and SEQ IDNO: 1096. The presence and/or amount of primer or probe that is annealedto said sample nucleic acid can be detected. In some embodiments, theprimers and/or probes can include the nucleic acid sequences of SEQ IDNO's 1090, 1091, 1096 and 2297, or the complements thereof.

Preferably, the primers and/or probes are placed in the same physicalenclosure.

In some embodiments, wherein at least one pair of primers is annealed tothe sample DNA, and wherein said primer pair include nucleic acidsequences that correspond to, or are fully complementary to, at least 10consecutive nucleotides of SEQ ID NO: 1090 and 1091 or SEQ ID NO: 1095and 1096, the methods also include a step of amplifying the sample DNAwith the annealed primer pair(s). For example, in some embodiments, theamplification step can include a method selected from the groupconsisting of:

(a) polymerase chain reaction (PCR),

(b) ligase chain reaction,

(c) nucleic acid sequence-based amplification,

(d) self-sustained sequence replication,

(e) strand displacement amplification,

(f) branched DNA signal amplification,

(g) nested PCR, and

(h) multiplex PCR.

Preferably, the amplification step includes a PCR amplification step.

In some embodiments that include an amplification step, the sample canalso be contacted with at least one probe that hybridizes to anamplification product produced from at least one of the primer pairs. Inpreferred embodiments, the probe includes at least 10 consecutivenucleotides of the sequence of SEQ ID NO: 2299 or 2300. For example, insome embodiments, the at least one probe includes the sequence of SEQ IDNO: 2299 or 2300.

It is an object of the present invention to provide a specific,ubiquitous and sensitive method using probes and/or amplificationprimers for determining the presence and/or amount of nucleic acids:

-   -   from any algal, archaeal, bacterial, fungal or parasitical        species in any sample suspected of containing said nucleic        acids, and optionally,    -   from specific microbial species or genera selected from the        group consisting of the species or genera listed in Table 4, and        optionally,    -   from an antimicrobial agents resistance gene selected from the        group consisting of the genes listed in Table 5, and optionally,    -   from a toxin gene selected from the group consisting of the        genes listed in Table 6,        -   wherein each of said nucleic acids or a variant or part            thereof comprises a selected target region hybridizable with            said probes or primers;        -   said method comprising the steps of contacting said sample            with said probes or primers and detecting the presence            and/or amount of hybridized probes or amplified products as            an indication of the presence and/or amount of said any            microbial species, specific microbial species or genus or            family or group and antimicrobial agents resistance gene            and/or toxin gene.

In a specific embodiment, a similar method directed to each specificmicrobial species or genus or family or group detection andidentification, antimicrobial agents resistance genes detection, toxingenes detection, and universal bacterial detection, separately, isprovided.

In a more specific embodiment, the method makes use of DNA fragmentsfrom conserved genes (proprietary sequences and sequences obtained frompublic databases), selected for their capacity to sensitively,specifically and ubiquitously detect the targeted algal, archaeal,bacterial, fungal or parasitical nucleic acids.

In a particularly preferred embodiment, oligonucleotides of at least 12nucleotides in length have been derived from the longer DNA fragments,and are used in the present method as probes or amplification primers.To be a good diagnostic candidate, an oligonucleotide of at least 12nucleotides should be capable of hybridizing with nucleic acids fromgiven microorganism(s), and with substantially all strains andrepresentatives of said microorganism(s); said oligonucleotide beingspecies-, or genus-, or family-, or group-specific or universal.

In another particularly preferred embodiment, oligonucleotides primersand probes of at least 12 nucleotides in length are designed for theirspecificity and ubiquity based upon analysis of our databases of tuf,atpD and recA sequences. These databases are generated using bothproprietary and public sequence information. Altogether, these databasesform a sequence repertory useful for the design of primers and probesfor the detection and identification of algal, archaeal, bacterial,fungal and parasitical microorganisms. The repertory can also besubdivided into subrepertories for sequence analysis leading to thedesign of various primers and probes.

The tuf, atpD and recA sequences databases as a product to assist thedesign of oligonucleotides primers and probes for the detection andidentification of algal, archaeal, bacterial, fungal and parasiticalmicroorganisms are also covered.

The proprietary oligonucleotides (probes and primers) are also anotherobject of this invention.

Diagnostic kits comprising probes or amplification primers such as thosefor the detection of a microbial species or genus or family or phylum orgroup selected from the following list consisting of Abiotrophiaadiacens, Acinetobacter baumanii, Actinomycetae, Bacteroides, Cytophagaand Flexibacter phylum, Bacteroides fragilis, Bordetella pertussis,Bordetella sp., Campylobacter jejuni and C. coli, Candida albicans,Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candidakrusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis,Candida zeylanoides, Candida sp., Chlamydia pneumoniae, Chlamydiatrachomatis, Clostridium sp., Corynebacterium sp., Crypococcusneoformans, Cryptococcus sp., Cryptosporidium parvum, Entamoeba sp.,Enterobacteriaceae group, Enterococcuscasseliflavus-flavescens-gallinarum group, Enterococcus faecalis,Enterococcus faecium, Enterococcus gallinarum, Enterococcus sp.,Escherichia coli and Shigella sp. group, Gemella sp., Giardia sp.,Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila,Legionella sp., Leishmania sp., Mycobacteriaceae family, Mycoplasmapneumoniae, Neisseria gonorrhoeae, platelets contaminants group (seeTable 14), Pseudomonas aeruginosa, Pseudomonads group, Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcussp., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcuspyogenes, Streptococcus sp., Trypanosoma brucei, Trypanosoma cruzi,Trypanosoma sp., Trypanosomatidae family, are also objects of thepresent invention.

Diagnostic kits further comprising probes or amplification primers forthe detection of an antimicrobial agents resistance gene selected fromthe group listed in Table 5 are also objects of this invention.

Diagnostic kits further comprising probes or amplification primers forthe detection of a toxin gene selected from the group listed in Table 6are also objects of this invention.

Diagnostic kits further comprising probes or amplification primers forthe detection of any other algal, archaeal, bacterial, fungal orparasitical species than those specifically listed herein, comprising ornot comprising those for the detection of the specific microbial speciesor genus or family or group listed above, and further comprising or notcomprising probes and primers for the antimicrobial agents resistancegenes listed in Table 5, and further comprising or not comprising probesand primers for the toxin genes listed in Table 6 are also objects ofthis invention.

In a preferred embodiment, such a kit allows for the separate or thesimultaneous detection and identification of the above-listed microbialspecies or genus or family or group; or universal detection of algae,archaea, bacteria, fungi or parasites; or antimicrobial agentsresistance genes; or toxin genes; or for the detection of anymicroorganism (algae, archaea, bacteria, fungi or parasites).

In the above methods and kits, probes and primers are not limited tonucleic acids and may include, but are not restricted to analogs ofnucleotides such as: inosine, 3-nitropyrrole nucleosides (Nichols etal., 1994, Nature 369:492-493), Linked Nucleic Acids (LNA) (Koskin etal., 1998, Tetrahedron 54:3607-3630), and Peptide Nucleic Acids (PNA)(Egholm et al., 1993, Nature 365:566-568).

In the above methods and kits, amplification reactions may include butare not restricted to: a) polymerase chain reaction (PCR), b) ligasechain reaction (LCR), c) nucleic acid sequence-based amplification(NASBA), d) self-sustained sequence replication (3 SR), e) stranddisplacement amplification (SDA), f) branched DNA signal amplification(bDNA), g) transcription-mediated amplification (TMA), h) cycling probetechnology (CPT), i) nested PCR, j) multiplex PCR, k) solid phaseamplification (SPA), l) nuclease dependent signal amplification (NDSA),m) rolling circle amplification technology (RCA), n) Anchored stranddisplacement amplification, o) Solid-phase (immobilized) rolling circleamplification.

In the above methods and kits, detection of the nucleic acids of targetgenes may include real-time or post-amplification technologies. Thesedetection technologies can include, but are not limited to, fluorescenceresonance energy transfer (FRET)-based methods such as adjacenthybridization to FRET probes (including probe-probe and probe-primermethods), TaqMan, Molecular Beacons, scorpions, nanoparticle probes andSunrise (Amplifluor). Other detection methods include target genesnucleic acids detection via immunological methods, solid phasehybridization methods on filters, chips or any other solid support,whether the hybridization is monitored by fluorescence,chemiluminescence, potentiometry, mass spectrometry, plasmon resonance,polarimetry, colorimetry, or scanometry. Sequencing, includingsequencing by dideoxy termination or sequencing by hybridization, e.g.sequencing using a DNA chip, is another possible method to detect andidentify the nucleic acids of target genes.

In a preferred embodiment, a PCR protocol is used for nucleic acidamplification, in diagnostic method as well as in method of constructionof a repertory of nucleic acids and deduced sequences.

In a particularly preferred embodiment, a PCR protocol is provided,comprising, an initial denaturation step of 1-3 minutes at 95° C.,followed by an amplification cycle including a denaturation step of onesecond at 95° C. and an annealing step of 30 seconds at 45-65° C.,without any time allowed specifically for the elongation step. This PCRprotocol has been standardized to be suitable for PCR reactions withmost selected primer pairs, which greatly facilitates the testingbecause each clinical sample can be tested with universal,species-specific, genus-specific, antimicrobial agents resistance geneand toxin gene PCR primers under uniform cycling conditions.Furthermore, various combinations of primer pairs may be used inmultiplex PCR assays.

It is also an object of the present invention that tuf, atpD and recAsequences could serve as drug targets and these sequences and means toobtain them revealed in the present invention can assist the screening,design and modeling of these drugs.

It is also an object of the present invention that tuf, atpD and recAsequences could serve for vaccine purposes and these sequences and meansto obtain them revealed in the present invention can assist thescreening, design and modeling of these vaccines.

We aim at developing a universal DNA-based test or kit to screen outrapidly samples which are free of algal, archaeal, bacterial, fungal orparasitical cells. This test could be used alone or combined with morespecific identification tests to detect and identify the above algaland/or archaeal and/or bacterial and/or fungal and/or parasiticalspecies and/or genera and/or family and/or group and to determinerapidly the bacterial resistance to antibiotics and/or presence ofbacterial toxins. Although the sequences from the selected antimicrobialagents resistance genes are available from public databases and havebeen used to develop DNA-based tests for their detection, our approachis unique because it represents a major improvement over currentdiagnostic methods based on bacterial cultures. Using an amplificationmethod for the simultaneous or independent or sequential microbialdetection-identification and antimicrobial resistance genes detection,there is no need for culturing the clinical sample prior to testing.Moreover, a modified PCR protocol has been developed to detect alltarget DNA sequences in approximately one hour under uniformamplification conditions. This procedure should save lives by optimizingtreatment, should diminish antimicrobial agents resistance because lessantibiotics will be prescribed, should reduce the use of broad spectrumantibiotics which are expensive, decrease overall health care costs bypreventing or shortening hospitalizations, and side effects of drugs,and decrease the time and costs associated with clinical laboratorytesting.

In another embodiment, sequence repertories and ways to obtain them forother gene targets are also an object of this invention, such is thecase for the hexA nucleic acids and/or sequences of Streptococci.

In yet another embodiment, for the detection of mutations associatedwith antibiotic resistance genes, we built repertories to distinguishbetween point mutations reflecting only gene diversity and pointmutations involved in resistance. Such repertories and ways to obtainthem for pbp1a, pbp2b and pbp2x genes of sensitive andpenicillin-resistant Streptoccoccus pneumoniae and also for gyrA andparC gene fragments from various bacterial species are also an object ofthe present invention.

The diagnostic kits, primers and probes mentioned above can be used toidentify algae, archaea, bacteria, fungi, parasites, antimicrobialagents resistance genes and toxin genes on any type of sample, whethersaid diagnostic kits, primers and probes are used for in vitro or insitu applications. The said samples may include but are not limited to:any clinical sample, any environment sample, any microbial culture, anymicrobial colony, any tissue, and any cell line.

It is also an object of the present invention that said diagnostic kits,primers and probes can be used alone or in conjunction with any otherassay suitable to identify microorganisms, including but not limited to:any immunoassay, any enzymatic assay, any biochemical assay, anylysotypic assay, any serological assay, any differential culture medium,any enrichment culture medium, any selective culture medium, anyspecific assay medium, any identification culture medium, anyenumeration culture medium, any cellular stain, any culture on specificcell lines, and any infectivity assay on animals.

In the methods and kits described herein below, the oligonucleotideprobes and amplification primers have been derived from larger sequences(i.e. DNA fragments of at least 100 base pairs). All DNA fragments havebeen obtained either from proprietary fragments or from publicdatabases. DNA fragments selected from public databases are newly usedin a method of detection according to the present invention, since theyhave been selected for their diagnostic potential.

In another embodiment, the amino acid sequences translated from therepertory of tuf, atpD and recA nucleic acids and/or sequences are alsoan object of the present invention.

It is clear to the individual skilled in the art that otheroligonucleotide sequences appropriate for (i) the universal detection ofalgae, archaea, bacteria, fungi or parasites, (ii) the detection andidentification of the above microbial species or genus or family orgroup, and (iii) the detection of antimicrobial agents resistance genes,and (iv) the detection of toxin genes, other than those listed in Tables39 to 41, 59 to 60, 70 to 74, 77 to 79, and 81 to 92 may also be derivedfrom the proprietary fragments or selected public database sequences.For example, the oligonucleotide primers or probes may be shorter orlonger than the ones chosen; they may also be selected anywhere else inthe proprietary DNA fragments or in the sequences selected from publicdatabases; they may be also variants of the same oligonucleotide. If thetarget DNA or a variant thereof hybridizes to a given oligonucleotide,or if the target DNA or a variant thereof can be amplified by a givenoligonucleotide PCR primer pair, the converse is also true; a giventarget DNA may hybridize to a variant oligonucleotide probe or beamplified by a variant oligonucleotide PCR primer. Alternatively, theoligonucleotides may be designed from any DNA fragment sequences for usein amplification methods other than PCR. Consequently, the core of thisinvention is the identification of universal, species-specific,genus-specific, family-specific, group-specific, resistancegene-specific, toxin gene-specific genomic or non-genomic DNA fragmentswhich are used as a source of specific and ubiquitous oligonucleotideprobes and/or amplification primers. Although the selection andevaluation of oligonucleotides suitable for diagnostic purposes requiresmuch effort, it is quite possible for the individual skilled in the artto derive, from the selected DNA fragments, oligonucleotides other thanthe ones listed in Tables 39 to 41, 59 to 60, 70 to 74, 77 to 79, and 81to 92 which are suitable for diagnostic purposes. When a proprietaryfragment or a public databases sequence is selected for its specificityand ubiquity, it increases the probability that subsets thereof willalso be specific and ubiquitous.

Since a high percentage of clinical specimens are negative for bacteria(Table 3), DNA fragments having a high potential for the selection ofuniversal oligonucleotide probes or primers were selected fromproprietary and public database sequences. The amplification primerswere selected from genes highly conserved in algae, archaea, bacteria,fungi and parasites, and are used to detect the presence of any algal,archaeal, bacterial, fungal or parasitical pathogen in clinicalspecimens in order to determine rapidly whether it is positive ornegative for algae, archaea, bacteria, fungi or parasites. The selectedgenes, designated tuf, fus, atpD and recA, encode respectively 2proteins (elongation factors Tu and G) involved in the translationalprocess during protein synthesis, a protein (beta subunit) responsiblefor the catalytic activity of proton pump ATPase and a proteinresponsible for the homologous recombination of genetic material. Thealignments of tuf, atpD and recA sequences used to derive the universalprimers include both proprietary and public database sequences. Theuniversal primer strategy allows the rapid screening of the numerousnegative clinical specimens (around 80% of the specimens received, seeTable 3) submitted for microbiological testing.

Table 4 provides a list of the archaeal, bacterial, fungal andparasitical species for which tuf and/or atpD and/or recA nucleic acidsand/or sequences are revealed in the present invention. Tables 5 and 6provide a list of antimicrobial agents resistance genes and toxin genesselected for diagnostic purposes. Table 7 provides the origin of tuf,atpD and recA nucleic acids and/or sequences listed in the sequencelisting. Tables 8-10 and 12-14 provide lists of species used to test thespecificity, ubiquity and sensitivity of some assays described in theexamples. Table 11 provides a list of microbial species for which tufand/or atpD and/or recA sequences are available in public databases.Table 15 lists the microorganisms identified by commercial systems.Tables 16-18 are part of Example 42, whereas Tables 19-20 are part ofExample 43. Tables 21-22 illustrate Example 44, whereas Tables 23-25illustrate Example 45.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate the principal subdivisions of the tuf and atpDsequences repertories, respectively. For the design of primers andprobes, depending on the needs, one may want to use the complete dataset illustrated on the top of the pyramid or use only a subsetillustrated by the different branching points. Smaller subdivisions,representing groups, families, genus and species, could even be made toextend to the bottom of the pyramid. Because the tuf and atpD sequencesare highly conserved and evolved with each species, the design ofprimers and probes does not need to include all the sequences within thedatabase or its subdivisions. As illustrated in Tables 42 to 58, 61 to69, 76 and 80, depending on the use, sequences from a limited number ofspecies can be carefully selected to represent: i) only the mainphylogenetic branches from which the intended probes and primers need tobe differentiating, and ii) only the species for which they need to bematching. However, for ubiquity purposes, and especially for primers andprobes identifying large groups of species (genus, family, group oruniversal, or sequencing primers), the more data is included into thesequence analysis, the better the probes and primers will be suitablefor each particular intended use. Similarly, for specificity purposes, alarger data set (or repertory) ensures optimal primers and probes designby reducing the chance of employing nonspecific oligonucleotides.

FIG. 3 illustrates the approach used to design specific amplificationprimers from fusA as well as from the region between the end of fusA andthe beginning of tuf in the streptomycin (str) operon (referred to asthe fusA-tuf intergenic spacer in Table 7). Shown is a schematicorganization of universal amplification primers (SEQ ID NOs. 1221-1229)in the str operon. Amplicon sizes are given in bases pairs. Drawing notto scale, as the fusA-tuf intergenic spacer size varies depending on thebacterial species. Indicated amplicon lengths are for E. coli.

FIGS. 4 to 6 are illustrations to Example 42, whereas FIGS. 7 to 10illustrate Example 43. FIGS. 11 and 12 illustrate Example 44.

FIGS. 4A and 4B: Abridged multiple amino acid sequence alignment of thepartial tuf gene products from selected species (SEQ ID NOS. 2303-2340)illustrated using the program Alscript. Residues highly conserved inbacteria are boxed in grey and gaps are represented with dots. Residuesin reverse print are unique to the enterococcal tufB as well as tostreptococcal and lactococcal tuf gene products. Numbering is based onE. coli EF-Tu and secondary structure elements of E. coli EF-Tu arerepresented by cylinders (α-helices) and arrows (β-strands).

FIG. 5. Distance matrix tree of bacterial EF-Tu based on amino acidsequence homology. The tree was constructed by the neighbor-joiningmethod. The tree was rooted using archeal and eukaryotic EF-1α genes asthe outgroup. The scale bar represents 5% changes in amino acidsequence, as determined by taking the sum of all of the horizontal linesconnecting two species.

FIG. 6. Southern hybridization of BglII/XbaI digested genomic DNAs ofsome enterococci (except for E. casseliflavus and E. gallinarum whosegenomic DNA was digested with BamHI/PvuII) using the tufA gene fragmentof E. faecium as probes. The sizes of hybridizing fragments are shown inkilobases. Strains tested are listed in Table 16.

FIG. 7. Pantoea and Tatumella species specific signature indel in atpDgenes. The nucleotide positions given are for E. coli atpD sequence(GenBank accession no. V00267). Numbering starts from the first base ofthe initiation codon.

FIGS. 8A and 8B: Trees based on sequence data from tuf (left side) andatpD (right side). The phylogenetic analysis was performed using theNeighbor-Joining method calculated using the Kimura two-parametermethod. The value on each branch indicates the occurrence (%) of thebranching order in 750 bootstrapped trees.

FIGS. 9a, 9b and 9c : Phylogenetic tree of members of the familyEnterobacteriaceae based on tuf (a), atpD (b), and 16S rDNA (c) genes.Trees were generated by neighbor-joining method calculated using theKimura two-parameter method. The value on each branch is the percentageof bootstrap replications supporting the branch. 750 bootstrapreplications were calculated.

FIGS. 10a, 10b and 10c : Plot of tuf distances versus 16S rDNA distances(a), atpD distances versus 16S rDNA distances (b), and atpD distancesversus tuf distances (c). Symbols: ◯, distances between pairs of strainsbelonging to the same species; ●, distances between E. coli strains andShigella strains; □, distances between pairs belonging to the samegenus; ▪, distances between pairs belonging to different genera; Δ,distances between pairs belonging to different families.

FIG. 11 depicts a multiple nucleic acid sequence alignment of the vanAgene from the indicated GenBank nucleotide accession numbers. Above thealignment of the GenBank sequences is a consensus sequence, derived fromthe alignment of the nucleic acid sequences below. Below the alignmentof the GenBank sequences and shaded in grey are the sequences ofoligonucleotides (SEQ ID NOs: 1090 and 1091) and the position of amolecular beacon probe (SEQ ID NO: 2299) that hybridizes to theamplification product of SEQ ID NOs: 1090 and 1091.

FIG. 12 depicts a multiple nucleic acid sequence alignment of the vanBgene from the indicated GenBank nucleotide accession numbers. Above thealignment of the GenBank sequences is a consensus sequence, derived fromthe alignment of the nucleic acid sequences below. Below the alignmentof the GenBank sequences and shaded in grey are the sequences ofoligonucleotides (SEQ ID NOs: 1096 and 2298) and the position of amolecular beacon probe (SEQ ID NO: 2300) that hybridizes to theamplification product of SEQ ID NOs: 1096 and 2298.

FIGS. 13A and 13B shows a graphical depictions of PCR amplificationcurves measured from reactions containing molecular beacon probes.Reactions contained 0, 0.5, 2.5, 5. 10, or 20 copies of vanA resistantE. faecium (FIG. 13A) or vanB resistant E. faecalis (FIG. 13B) templateDNA, as well as 3.5 copies of internal control DNA. Molecular beaconprobes (SEQ ID NO: 2299 and 2300) were added to each reaction and thefluorescence of the reactions was measured (FIGS. 13A and 13B,respectively). SEQ ID NO: 2299 is labeled with FAM. SEQ ID NO: 2300 islabeled with Texas Red. SEQ ID NO: 2301 is labeled with TET.

FIGS. 14A and 14B shows an agarose gel of the DNA amplification productsfrom PCR using the template DNA sources listed in Table 29. The numbersabove the lanes correspond to the numbers in Table 29.

FIGS. 15A and 15B show an agarose gel of the DNA amplification productsfrom PCR using template DNA sources listed in Table 30. The numbersabove the lanes correspond to the numbers in Table 30.

FIGS. 16A and 16B show an agarose gel of the DNA amplification productsfrom PCR using template DNA sources listed in Table 31. The numbersabove the lanes correspond to the numbers in Table 31.

FIG. 17 shows an agarose gel of the DNA amplification products from PCRusing template DNA sources listed in Table 32. The numbers above thelanes correspond to the numbers in Table 32.

FIGS. 18A and 18B show the fluorescence signal readout obtained in theFAM channel when vanA template (FIG. 18A) or non-specific template (FIG.18B) DNA was used in PCR according to Example 23.

FIGS. 19A and 19B show the fluorescence signal readout obtained in theTET channel when internal control (IC) template (FIG. 19A) ornon-specific template (FIG. 19B) DNA was used in PCR according toExample 23

FIGS. 20A and 20B show the fluorescence signal readout obtained in theTexas Red channel when vanB template (FIG. 20A) or non-specific template(FIG. 20B) DNA was used in the PCR according to Example 23.

FIG. 21 shows the fluorescent signal readout obtained in the vanR assayfor a vanA positive clinical specimen. The top panel shows thefluorescent readout from the FAM channel, and the bottom panel shows thefluorescent readout in the Texas Red channel, designed to detect thevanB probe.

FIG. 22 shows the fluorescent signal readout obtained in the vanR assayfor a clinical specimen that is both vanA and vanB positive. The toppanel shows the readout from the FAM channel (vanA) and the bottom panela shows the readout from the Texas Red channel (vanB).

FIG. 23 shows the fluorescent signal readout obtained in the vanR assayfor a clinical specimen that is vanB positive. The top panel shows theFAM channel (vanA) and the bottom channel shows the fluorescent readoutfrom the Texas Red channel (vanB).

FIG. 24 shows an agarose gel of the DNA amplification products from PCRusing the template DNA sources listed in Table 36. The numbers above thelanes correspond to the numbers in Table 36.

FIG. 25 shows an agarose gel of the DNA amplification products from PCRusing the template DNA sources listed in Table 37. The numbers above thelanes correspond to the numbers in Table 37.

FIG. 26 shows nucleotide and amino acid sequence identities of EF-Tubetween different enterococci and other low G+C gram-positive bacteria.The upper right triangle represents the deduced amino acid sequenceidentities of gram-positive bacterial EF-Tu, while the lower lefttriangle represents the DNA sequence identities of the corresponding tufgenes. The sequence identities between different enterococcal tufA genesare shown in the boxed area encompassing rows 1-16 and columns 1-16,while those between enterococcal tufB genes are shown in the boxed areaencompassing rows 18-28 and columns 18-28.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present inventors reasoned that comparing the published Haemophilusinfluenzae and Mycoplasma genitalium genomes and searching for conservedgenes could provide targets to develop useful diagnostic primers andprobes. This sequence comparison is highly informative as these twobacteria are distantly related and most genes present in the minimalgenome of M. genitalium are likely to be present in every bacterium.Therefore genes conserved between these two bacteria are likely to beconserved in all other bacteria.

Following the genomic comparison, it was found that severalprotein-coding genes were conserved in evolution. Highly conservedproteins included the translation elongation factors G (EF-G) and Tu(EF-Tu) and the β subunit of F₀F₁ type ATP-synthase, and to a lesserextent, the RecA recombinase. These four proteins coding genes wereselected amongst the 20 most conserved genes on the basis that they allpossess at least two highly conserved regions suitable for the design ofuniversal amplification and sequencing primers. Moreover, within thefragment amplified by these primers, highly conserved and more variableregions are also present hence suggesting it might be possible torapidly obtain sequence information from various microbial species todesign universal as well as species-, genus-, family-, or group-specificprimers and probes of potential use for the detection and identificationand/or quantification of microorganisms.

Translation elongation factors are members of a family of GTP-bindingproteins which intervene in the interactions of tRNA molecules with theribosome machinery during essential steps of protein synthesis. The roleof elongation factor Tu is to facilitate the binding of aminoacylatedtRNA molecules to the A site of the ribosome. The eukaryotic, archaeal(archaebacterial) and algal homolog of EF-Tu is called elongation factor1 alpha (EF-□α). All protein synthesis factors originated from a commonancestor via gene duplications and fusions (Cousineau et al., 1997, J.Mol. Evol. 45:661-670). In particular, elongation factor G (EF-G),although having a functional role in promoting the translocation ofaminoacyl-tRNA molecules from the A site to the P site of the ribosome,shares sequence homologies with EF-Tu and is thought to have arisen fromthe duplication and fusion of an ancestor of the EF-Tu gene.

In addition, EF-Tu is known to be the target for antibiotics belongingto the elfamycin's group as well as to other structural classes (Anborghand Parmeggiani, 1991, EMBO J. 10:779-784; Luiten et al., 1992, Europeanpatent application serial No. EP 0 466 251 A1). EF-G for its part, isthe target of the antibiotic fusidic acid. In addition to its crucialactivities in translation, EF-Tu has chaperone-like functions in proteinfolding, protection against heat denaturation of proteins andinteractions with unfolded proteins (Caldas et al., 1998, J. Biol. Chem273:11478-11482). Interestingly, a form of the EF-Tu protein has beenidentified as a dominant component of the periplasm of Neisseriagonorrhoeae (Porcella et al., 1996, Microbiology 142:2481-2489), hencesuggesting that at least in some bacterial species, EF-Tu might be anantigen with vaccine potential.

F₀F₁ type ATP-synthase belongs to a superfamily of proton-translocatingATPases divided in three major families: P, V and F (Nelson and Taiz,1989, TIBS 14:113-116). P-ATPases (or E₁-E₂ type) operate via aphosphorylated intermediate and are not evolutionarily related to theother two families. V-ATPases (or V₀V₁ type) are present on the vacuolarand other endomembranes of eukaryotes, on the plasma membrane of archaea(archaebacteria) and algae, and also on the plasma membrane of someeubacteria especially species belonging to the order Spirochaetales aswell as to the Chlamydiaceae and Deinococcaceae families. F-ATPases (orF₀F₁ type) are found on the plasma membrane of most eubacteria, on theinner membrane of mitochondria and on the thylakoid membrane ofchloroplasts. They function mainly in ATP synthesis. They are largemultimeric enzymes sharing numerous structural and functional featureswith the V-ATPases. F and V-type ATPases have diverged from a commonancestor in an event preceding the appearance of eukaryotes. The βsubunit of the F-ATPases is the catalytic subunit and it possesses lowbut significant sequence homologies with the catalytic A subunit ofV-ATPases.

The translation elongation factors EF-Tu, EF-G and EF-1α□ and thecatalytic subunit of F or V-types ATP-synthase, are highly conservedproteins sometimes used for phylogenetic analysis and their genes arealso known to be highly conserved (Iwabe et al., 1989, Proc. Natl. Acad.Sci. USA 86:9355-9359, Gogarten et al., 1989, Proc. Natl. Acad. Sci. USA86:6661-6665, Ludwig et al., 1993, Antonie van Leeuwenhoek 64:285-305).A recent BLAST (Altschul et al., 1997, J. Mol. Biol. 215:403-410) searchperformed by the present inventors on the GenBank, European MolecularBiology Laboratory (EMBL), DNA Database of Japan (DDBJ) and specificgenome project databases indicated that throughout bacteria, the EF-Tuand the β subunit of F₀F₁ type ATP-synthase genes may be more conservedthan other genes that are well conserved between H. influenzae and M.genitalium.

The RecA recombinase is a multifunctional protein encoded by the recAgene. It plays a central role in homologous recombination, it iscritical for the repair of DNA damage and it is involved in theregulation of the SOS system by promoting the proteolytic digestion ofthe LexA repressor. It is highly conserved in bacteria and could serveas a useful genetic marker to reconstruct bacterial phylogeny (Millerand Kokjohn, 1990, Annu. Rev. Microbiol. 44:365-394). Although RecApossesses some highly conserved sequence segments that we used to designuniversal primers aimed at sequencing the recA fragments, it is clearlynot as well conserved EF-G, EF-Tu and β subunit of F₀F₁ typeATP-synthase. Hence, RecA may not be optimal for universal detection ofbacteria with high sensitivity but it was chosen because preliminarydata indicated that EF-G, EF-Tu and β subunit of F₀F₁ type ATP-synthasemay sometimes be too closely related to find specific primer pairs thatcould discriminate between certain very closely related species andgenera. While RecA, EF-G, EF-Tu and β subunit of F₀F₁ type ATP-synthasegenes, possesses highly conserved regions suitable for the design ofuniversal sequencing primers, the less conserved region between primersshould be divergent enough to allow species-specific and genus-specificprimers in those cases.

Thus, as targets to design primers and probes for the genetic detectionof microorganisms, the present inventors have focused on the genesencoding these four proteins: tuf, the gene for elongation factor Tu(EF-Tu); fus, the gene for the elongation factor G (EF-G); atpD, thegene for β subunit of F₀F₁ type ATP-synthase; and recA, the geneencoding the RecA recombinase. In several bacterial genomes tuf is oftenfound in two highly similar duplicated copies named tufA and tufB (Filerand Furano, 1981, J. Bacteriol. 148:1006-1011, Sela et al., 1989, J.Bacteriol. 171:581-584). In some particular cases, more divergent copiesof the tuf genes can exist in some bacterial species such as someactinomycetes (Luiten et al. European patent application publication No.EP 0 446 251 A1; Vijgenboom et al., 1994, Microbiology 140:983-998) and,as revealed as part of this invention, in several enterococcal species.In several bacterial species, tuf is organized in an operon with itshomolog gene for the elongation factor G (EF-G) encoded by the fusA gene(FIG. 3). This operon is often named the str operon. The tuf, fus, atpDand recA genes were chosen as they are well conserved in evolution andhave highly conserved stretches as well as more variable segments.Moreover, these four genes have eukaryotic orthologs which are describedin the present invention as targets to identify fungi and parasites. Theeukaryotic homolog of elongation factor Tu is called elongation factor1-alpha (EF-1α) (gene name: tef, tef1, ef1, ef-1 or EF-1). In fungi, thegene for EF-1α occurs sometimes in two or more highly similar duplicatedcopies (often named tef1, tef2, tef3 . . . ). In addition, eukaryoteshave a copy of elongation factor Tu which is originating from theirorganelle genome ancestry (gene name: tuf1, tufM or tufA). For thepurpose of the current invention, the genes for these four functionallyand evolutionarily linked elon-gation factors (bacterial EF-Tu and EF-G,eukaryotic EF-1α, and organellar EF-Tu) will hereafter be designated as«tuf nucleic acids and/or sequences». The eukaryotic (mitochondrial)F₀F₁ type ATP-synthase beta subunit gene is named atp2 in yeast. For thepurpose of the current invention, the genes of catalytic sub-unit ofeither F or V-type ATP-synthase will hereafter be designated as «atpDnucleic acids and/or sequences». The eukaryotic homologs of RecA aredistributed in two families, typified by the Rad51 and Dmc1 proteins.Archaeal homologs of RecA are called RadA. For the purpose of thecurrent invention, the genes corres-ponding to the latter proteins willhereafter be designated as «recA nucleic acids and/or sequences».

In the description of this invention, the terms «nucleic acids» and«sequences» might be used interchangeably. However, «nucleic acids» arechemical entities while «sequences» are the pieces of informationderived from (inherent to) these «nucleic acids». Both nucleic acids andsequences are equiva-lently valuable sources of information for thematter pertaining to this invention.

Analysis of multiple sequence alignments of tuf and atpD sequencespermitted the design of oligonucleotide primers (and probes) capable ofamplifying (or hybridizing to) segments of tuf (and/or fus) and atpDgenes from a wide variety of bacterial species (see Examples 1 to 4, 24and 26, and Table 7). Sequencing and amplification primer pairs for tufnucleic acids and/or sequences are listed in Table 39 and hybridizationprobes are listed in Tables 41 and 85. Sequencing and amplificationprimer pairs for atpD nucleic acids and/or sequences are listed in Table40. Analysis of the main subdivisions of tuf and atpD sequences (seeFIGS. 1 and 2) permitted to design sequencing primers amplifyingspecifically each of these subdivisions. It should be noted that thesesequencing primers could also be used as universal primers. However,since some of these sequencing primers include several variable sequence(degenerated) positions, their sensitivity could be lower than that ofuniversal primers developed for diagnostic purposes. Furthersubdivisions could be done on the basis of the various phyla where thesegenes are encountered.

Similarly, analysis of multiple sequence alignments of recA sequencespresent in the public databases permitted the design of oligonucleotideprimers capable of amplifying segments of recA genes from a wide varietyof bacterial species. Sequencing and amplification primer pairs for recAsequences are listed in Table 59. The main subdivisions of recA nucleicacids and/or sequences comprise recA, radA, rad51 and dmc1. Furthersubdivisions could be done on the basis of the various phyla where thesegenes are encountered.

The present inventor's strategy is to get as much sequence datainformation from the four conserved genes (tuf, fus, atpD and recA).This ensemble of sequence data forming a repertory (with subrepertoriescorresponding to each target gene and their main sequence subdivisions)and then using the sequence information of the sequence repertory (orsubrepertories) to design primer pairs that could permit eitheruniversal detection of algae or archaea or bacteria or fungi orparasites, detection of a family or group of microorganism (e.g.Enterobacteriaceae), detection of a genus (e.g. Streptococcus) orfinally a specific species (e.g. Staphylococcus aureus). It should benoted that for the purpose of the present invention a group ofmicroorganisms is defined depending on the needs of the particulardiagnostic test. It does not need to respect a particular taxonomicalgrouping or phylum. See Example 12 where primers were designed toamplify a group a bacteria consisting of the 17 major bacterial speciesencountered as contaminants of platelet concentrates. Also remark thatin that Example, the primers are not only able to sensitively andrapidly detect at least the 17 important bacterial species, but couldalso detect other species as well, as shown in Table 14. In thesecircumstances the primers shown in Example 12 are considered universalfor platelet-contaminating bacteria. To develop an assay specific forthe latter, one or more primers or probes specific to each species couldbe designed. Another example of primers and/or probes for groupdetection is given by the Pseudomonad group primers. These primers weredesigned based upon alignment of tuf sequences from real Pseudomonasspecies as well as from former Pseudomonas species such asStenotrophomonas maltophilia. The resulting primers are able to amplifyall Pseudomonas species tested as well as several species belonging todifferent genera, hence as being specific for a group includingPseudomonas and other species, we defined that group as Pseudomonads, asseveral members were former Pseudomonas.

For certain applications, it may be possible to develop a universal,group, family or genus-specific reaction and to proceed to speciesidentification using sequence information within the amplicon to designspecies-specific internal probes or primers, or alternatively, toproceed directly by sequencing the amplicon. The various strategies willbe discussed further below.

The ensembles formed by public and proprietary tuf, atpD and recAnucleic acids and/or sequences are used in a novel fashion so theyconstitute three databases containing useful information for theidentification of microorganisms.

Sequence repertories of other gene targets were also built to solve somespecific identification problems especially for microbial speciesgenetically very similar to each other such as E. coli and Shigella (seeExample 23). Based on tuf, atpD and recA sequences, Streptococcuspneumoniae is very difficult to differentiate from the closely relatedspecies S. oralis and S. mitis. Therefore, we elected to built asequence repertory from hexA sequences (Example 19), a gene much morevariable than our highly conserved tuf, atpD and recA nucleic acidsand/or sequences.

For the detection of mutations associated with antibiotic resistancegenes, we also built repertories to distinguish between point mutationsreflecting only gene diversity and point mutations involved inresistance. This was done for pbp1a, pbp2b and pbp2x genes ofpenicillin-resistant and sensitive Streptoccoccus pneumoniae (Example18) and also for gyrA and parC gene fragments of various bacterialspecies for which quinolone resistance is important to monitor.

Oligonucleotide Primers and Probes Design and Synthesis

The tuf, fus, atpD and recA DNA fragments sequenced by us and/orselected from public databases (GenBank and EMBL) were used to designoligonucleotides primers and probes for diagnostic purposes. Multiplesequence alignments were made using subsets of the tuf or atpD or recAsequences repertory. Subsets were chosen to encompass as much aspossible of the targeted microorganism(s) DNA sequence data and alsoinclude sequence data from phylogenetically related microorganisms fromwhich the targeted microorganism(s) should be distinguished. Regionssuitable for primers and probes should be conserved for the targetedmicroorganism(s) and divergent for the microorganisms from which thetargeted microorganism(s) should be distinguished. The large amount oftuf or atpD or recA sequences data in our repertory permits to reducetrial and errors in obtaining specific and ubiquitous primers andprobes. We also relied on the corresponding peptide sequences of tuf,fus, atpD and recA nucleic acids and/or sequences to facilitate theidentification of regions suitable for primers and probes design. Aspart of the design rules, all oligonucleotides (probes for hybridizationand primers for DNA amplification by PCR) were evaluated for theirsuitability for hybridization or PCR amplification by computer analysisusing standard programs (i.e. the Genetics Computer Group (GCG) programsand the primer analysis software Oligo™ 5.0). The potential suitabilityof the PCR primer pairs was also evaluated prior to the synthesis byverifying the absence of unwanted features such as long stretches of onenucleotide and a high proportion of G or C residues at the 3′ end(Persing et al., 1993, Diagnostic Molecular Microbiology: Principles andApplications, American Society for Microbiology, Washington, D.C.).Oligonucleotide probes and amplification primers were synthesized usingan automated DNA synthesizer (Perkin-Elmer Corp., Applied BiosystemsDivision).

The oligonucleotide sequence of primers or probes may be derived fromeither strand of the duplex DNA. The primers or probes may consist ofthe bases A, G, C, or T or analogs and they may be degenerated at one ormore chosen nucleotide position(s). The primers or probes may be of anysuitable length and may be selected anywhere within the DNA sequencesfrom proprietary fragments or from selected database sequences which aresuitable for (i) the universal detection of algae or archaea or bacteriaor fungi or parasites, (ii) the species-specific detection andidentification of any microorganism, including but not limited to:Abiotrophia adiacens, Bacteroides fragilis, Bordetella pertussis,Candida albicans, Candida dubliniensis, Candida glabrata, Candidaguilliermondii, Candida krusei, Candida lusitaniae, Candidaparapsilosis, Candida tropicalis, Candida zeylanoides, Campylobacterjejuni and C. coli, Chlamydia pneumoniae, Chlamydia trachomatis,Cryptococcus neoformans, Cryptosporidium parvum, Enterococcus faecalis,Enterococcus faecium, Enterococcus gallinarum, Escherichia coli,Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Pseudomonas aeruginosa, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus saprophyticus, Streptococcus agalactiae,Streptococcus pneumoniae, Trypanosoma brucei, Trypanosoma cruzi, (iii)the genus-specific detection of Bordetella species, Candida species,Clostridium species, Corynebacterium species, Cryptococcus species,Entamoeba species, Enterococcus species, Gemella species, Giardiaspecies, Legionella species, Leishmania species, Staphylococcus species,Streptococcus species, Trypanosoma species, (iv) the family-specificdetection of Enterobacteriaceae family members, Mycobacteriaceae familymembers, Trypanosomatidae family members, (v) the detection ofEnterococcus casseliflavus-flavescens-gallinarum group, Enterococcus,Gemella and Abiotrophia adiacens group, Pseudomonads extended group,Platelet-contaminating bacteria group, (vi) the detection of clinicallyimportant antimicrobial agents resistance genes listed in Table 5, (vii)the detection of clinically important toxin genes listed in Table 6.

Variants for a given target microbial gene are naturally occurring andare attributable to sequence variation within that gene during evolution(Watson et al., 1987, Molecular Biology of the Gene, 4^(th) ed., TheBenjamin/Cummings Publishing Company, Menlo Park, Calif.; Lewin, 1989,Genes IV, John Wiley & Sons, New York, N.Y.). For example, differentstrains of the same microbial species may have a single or morenucleotide variation(s) at the oligonucleotide hybridization site. Theperson skilled in the art is well aware of the existence of variantalgal, archaeal, bacterial, fungal or parasitical DNA nucleic acidsand/or sequences for a specific gene and that the frequency of sequencevariations depends on the selective pressure during evolution on a givengene product. The detection of a variant sequence for a region betweentwo PCR primers may be demonstrated by sequencing the amplificationproduct. In order to show the presence of sequence variants at theprimer hybridization site, one has to amplify a larger DNA target withPCR primers outside that hybridization site. Sequencing of this largerfragment will allow the detection of sequence variation at this site. Asimilar strategy may be applied to show variants at the hybridizationsite of a probe. Insofar as the divergence of the target nucleic acidsand/or sequences or a part thereof does not affect the specificity andubiquity of the amplification primers or probes, variant microbial DNAis under the scope of this invention. Variants of the selected primersor probes may also be used to amplify or hybridize to a variant DNA.

Sequencing of tuf Nucleic Acids and/or Sequences from a Variety ofArchaeal, Bacterial, Fungal and Parasitical Species

The nucleotide sequence of a portion of tuf nucleic acids and/orsequences was determined for a variety of archaeal, bacterial, fungaland parasitical species. The amplification primers (SEQ ID NOs. 664 and697), which amplify a tuf gene portion of approximately 890 bp, wereused along with newly designed sequencing primer pairs (See Table 39 forthe sequencing primers for tuf nucleic acids and/or sequences). Mostprimer pairs can amplify different copies of tuf genes (tufA and tufB).This is not surprising since it is known that for several bacterialspecies these two genes are nearly identical. For example, the entiretufA and tufB genes from E. coli differ at only 13 nucleotide positions(Neidhardt et al., 1996, Escherichia coli and Salmonella: Cellular andMolecular Biology, 2^(nd) ed., American Society for Microbiology Press,Washington, D.C.). Similarly, some fungi are known to have two nearlyidentical copies of tuf nucleic acids and/or sequences (EF-1□). Theseamplification primers are degenerated at several nucleotide positionsand contain inosines in order to allow the amplification of a wide rangeof tuf nucleic acids and/or sequences. The strategy used to select theseamplification primers is similar to that illustrated in Table 39 for theselection of universal primers. The tuf sequencing primers evensometimes amplified highly divergent copies of tuf genes (tufC) asillustrated in the case of some enterococcal species (SEQ ID NOs.: 73,75, 76, 614 to 618, 621 and 987 to 989). To prove this, we havedetermined the enterococcal tuf nucleic acids and/or sequences from PCRamplicons cloned into a plasmid vector. Using the sequence data from thecloned amplicons, we designed new sequencing primers specific to thedivergent (tufC) copy of enterococci (SEQ ID NOs.: 658-659 and 661) andthen sequenced directly the tufC amplicons. The amplification primers(SEQ ID NOs.: 543, 556, 557, 643-645, 660, 664, 694, 696 and 697) couldbe used to amplify the tuf nucleic acids and/or sequences from anybacterial species. The amplification primers (SEQ ID NOs.: 558, 559,560, 653, 654, 655, 813, 815, 1974-1984, 1999-2003) could be used toamplify the tuf (EF-1□□□ genes from any fungal and/or parasiticalspecies. The amplification primers SEQ ID NOs. 1221-1228 could be usedto amplify bacterial tuf nucleic acids and/or sequences of the EF-Gsubdivision (fusA) (FIG. 3). The amplification primers SEQ ID NOs. 1224,and 1227-1229 could be used to amplify bacterial tuf nucleic acidsand/or sequences comprising the end of EF-G (fusA) and the beginning ofEF-Tu (tuf), including the intergenic region, as shown in FIG. 3.

Most tuf fragments to be sequenced were amplified using the followingamplification protocol: One μl of cell suspension (or of purifiedgenomic DNA 0.1-100 ng/μ1) was transferred directly to 19 μl of a PCRreaction mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl(pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 1 μM of each of the 2primers, 200 μM of each of the four dNTPs, 0.5 unit of Taq DNApolymerase (Promega Corp., Madison, Wis.). PCR reactions were subjectedto cycling using a PTC-200 thermal cycler (MJ Research Inc., Watertown,Mass.) as follows: 3 min at 94-96° C. followed by 30-45 cycles of 1 minat 95° C. for the denaturation step, 1 min at 50-55° C. for theannealing step and 1 min at 72° C. for the extension step. Subsequently,twenty microliters of the PCR-amplified mixture were resolved byelectrophoresis in a 1.5% agarose gel. The amplicons were thenvisualized by staining with methylene blue (Flores et al., 1992,Biotechniques, 13:203-205). The size of the amplification products wasestimated by comparison with a 100-bp molecular weight ladder. The bandcorresponding to the specific amplification product was excised from theagarose gel and purified using the QIAquick™ gel extraction kit (QIAGENInc., Chatsworth, Calif.). The gel-purified DNA fragment was then useddirectly in the sequencing protocol. Both strands of the tuf genesamplification product were sequenced by the dideoxynucleotide chaintermination sequencing method by using an Applied Biosystems automatedDNA sequencer (model 377) with their Big Dye™ Terminator CycleSequencing Ready Reaction Kit (Applied Biosystems, Foster City, Calif.).The sequencing reactions were performed by using the same amplificationprimers and 10 ng/100 bp of the gel-purified amplicon per reaction. Forthe sequencing of long amplicons such as those of eukaryotic tuf(EF-1□□□ nucleic acids and/or sequences, we designed internal sequencingprimers (SEQ ID NOs.: 654, 655 and 813) to be able to obtain sequencedata on both strands for most of the fragment length. In order to ensurethat the determined sequence did not contain errors attributable to thesequencing of PCR artifacts, we have sequenced two preparations of thegel-purified tuf amplification product originating from two independentPCR amplifications. For most target microbial species, the sequencesdetermined for both amplicon preparations were identical. In case ofdiscrepancies, amplicons from a third independent PCR amplification weresequenced. Furthermore, the sequences of both strands were 100%complementary thereby confirming the high accuracy of the determinedsequence. The tuf nucleic acids and/or sequences determined using theabove strategy are described in the Sequence Listing. Table 7 gives theoriginating microbial species and the source for each tuf sequence inthe Sequence Listing.

The alignment of the tuf sequences determined by us or selected fromdatabases revealed clearly that the length of the sequenced portion ofthe tuf genes is variable. There may be insertions or deletions ofseveral amino acids. In addition, in several fungi introns wereobserved. Intron nucleic acids and/or sequences are part of tuf nucleicacids and/or sequences and could be useful in the design ofspecies-specific primers and probes. This explains why the size of thesequenced tuf amplification products was variable from one fungalspecies to another. Consequently, the nucleotide positions indicated ontop of each of Tables 42 to 58, 61 to 69, 76 and 80 do not correspondfor sequences having insertions or deletions.

It should also be noted that the various tuf nucleic acids and/orsequences determined by us occasionally contain base ambiguities. Thesedegenerated nucleotides correspond to sequence variations between tufAand tufB genes (or copies of the EF-G subdivision of tuf nucleic acidsand/or sequences, or copies of EF-1□□ subdivision of tuf nucleic acidsand/or sequences for fungi and parasites) because the amplificationprimers amplify both tuf genes. These nucleotide variations were notattributable to nucleotide misincorporations by the Taq DNA polymerasebecause the sequence of both strands was identical and also because thesequences determined with both preparations of the gel-purified tufamplicons obtained from two independent PCR amplifications wereidentical.

The Selection of Amplification Primers from tuf Nucleic Acids and/orSequences

The tuf sequences determined by us or selected from public databaseswere used to select PCR primers for universal detection of bacteria, aswell as for genus-specific, species-specific family-specific orgroup-specific detection and identification. The strategy used to selectthese PCR primers was based on the analysis of multiple sequencealignments of various tuf sequences. For more details about theselection of PCR primers from tuf sequences please refer to Examples 5,7-14, 17, 22, 24, 28, 30-31, 33, 36, and 38-40, and to Tables 44-47,49-57 and 63.

Sequencing of atpD and recA Nucleic Acids and/or Sequences from aVariety of Archaeal, Bacterial, Fungal and Parasitical Species

The method used to obtain atpD and recA nucleic acids and/or sequencesis similar to that described above for tuf nucleic acids and/orsequences.

The Selection of Amplification Primers from atpD or recA Nucleic Acidsand/or Sequences

The comparison of the nucleotide sequence for the atpD or recA genesfrom various archaeal, bacterial, fungal and parasitical species allowedthe selection of PCR primers (refer to Examples 6, 13, 29, 34 and 37,and to Tables 42, 43, 48, and 58).

DNA Amplification

For DNA amplification by the widely used PCR (polymerase chain reaction)method, primer pairs were derived from proprietary DNA fragments or fromdatabase sequences. Prior to synthesis, the potential primer pairs wereanalyzed by using the Oligo™ 5.0 software to verify that they were goodcandidates for PCR amplification.

During DNA amplification by PCR, two oligonucleotide primers bindingrespectively to each strand of the heat-denatured target DNA from themicrobial genome are used to amplify exponentially in vitro the targetDNA by successive thermal cycles allowing denaturation of the DNA,annealing of the primers and synthesis of new targets at each cycle(Persing et al, 1993, Diagnostic Molecular Microbiology: Principles andApplications, American Society for Microbiology, Washington, D.C.).

Briefly, the PCR protocols were as follows: Treated clinical specimensor standardized bacterial or fungal or parasitical suspensions (seebelow) or purified genomic DNA from bacteria, fungi or parasites wereamplified in a 20 μl PCR reaction mixture. Each PCR reaction contained50 mM KCl, 10 mM Tris-HCl (pH 9.0), 2.5 mM MgCl₂, 0.4 μM of each primer,200 μM of each of the four dNTPs and 0.5 unit of Taq DNA polymerase(Promega) combined with the TaqStart™ antibody (Clontech LaboratoriesInc., Palo Alto, Calif.). The TaqStart™ antibody, which is aneutralizing monoclonal antibody to Taq DNA polymerase, was added to allPCR reactions to enhance the specificity and the sensitivity of theamplifications (Kellogg et al., 1994, Biotechniques 16:1134-1137). Thetreatment of the clinical specimens varies with the type of specimentested, since the composition and the sensitivity level required aredifferent for each specimen type. It consists in a rapid protocol tolyse the microbial cells and eliminate or neutralize PCR inhibitors. Foramplification from bacterial or fungal or parasitical cultures or frompurified genomic DNA, the samples were added directly to the PCRamplification mixture without any pre-treatment step. An internalcontrol was derived from sequences not found in the targetmicroorganisms or in the human genome. The internal control wasintegrated into all amplification reactions to verify the efficiency ofthe PCR assays and to ensure that significant PCR inhibition was absent.Alternatively, an internal control derived from rRNA was also useful tomonitor the efficiency of microbial lysis protocols.

PCR reactions were then subjected to thermal cycling (3 min at 94-96° C.followed by 30 cycles of 1 second at 95° C. for the denaturation stepand 30 seconds at 50-65° C. for the annealing-extension step) using aPTC-200 thermal cycler (MJ Research Inc.). The number of cyclesperformed for the PCR assays varies according to the sensitivity levelrequired. For example, the sensitivity level required for microbialdetection directly from clinical specimens is higher for blood specimensthan for urine specimens because the concentration of microorganismsassociated with a septicemia can be much lower than that associated witha urinary tract infection. Consequently, more sensitive PCR assayshaving more thermal cycles are probably required for direct detectionfrom blood specimens. Similarly, PCR assays performed directly frombacterial or fungal or parasitical cultures may be less sensitive thanPCR assays performed directly from clinical specimens because the numberof target organisms is normally much lower in clinical specimens than inmicrobial cultures.

The person skilled in the art of DNA amplification knows the existenceof other rapid amplification procedures such as ligase chain reaction(LCR), transcription-mediated amplification (TMA), self-sustainedsequence replication (3SR), nucleic acid sequence-based amplification(NASBA), strand displacement amplification (SDA), branched DNA (bDNA),cycling probe technology (CPT), solid phase amplification (SPA), rollingcircle amplification technology (RCA), solid phase RCA, anchored SDA andnuclease dependent signal amplification (NDSA) (Lee et al., 1997,Nucleic Acid Amplification Technologies: Application to DiseaseDiagnosis, Eaton Publishing, Boston, Mass.; Persing et al., 1993,Diagnostic Molecular Microbiology: Principles and Applications, AmericanSociety for Microbiology, Washington, D.C.; Westin et al., 2000, Nat.Biotechnol. 18:199-204). The scope of this invention is not limited tothe use of amplification by PCR, but rather includes the use of anyrapid nucleic acid amplification method or any other procedure which maybe used to increase the sensitivity and/or the rapidity of nucleicacid-based diagnostic tests. The scope of the present invention alsocovers the use of any nucleic acids amplification and detectiontechnology including real-time or post-amplification detectiontechnologies, any amplification technology combined with detection, anyhybridization nucleic acid chips or arrays technologies, anyamplification chips or combination of amplification and hybridizationchips technologies. Detection and identification by any sequencingmethod is also under the scope of the present invention.

Any oligonucleotide suitable for the amplification of nucleic acids byapproaches other than PCR or for DNA hybridization which are derivedfrom the species-specific, genus-specific and universal DNA fragments aswell as from selected antimicrobial agents resistance or toxin genesequences included in this document are also under the scope of thisinvention.

Detection of Amplification Products

Classically, detection of amplification is performed by standardethidium bromide-stained agarose gel electrophoresis. It is clear thatother methods for the detection of specific amplification products,which may be faster and more practical for routine diagnosis, may beused. Such methods may be based on the detection of fluorescence afteror during amplification. One simple method for monitoring amplified DNAis to measure its rate of formation by measuring the increase influorescence of intercalating agents such as ethidium bromide or SYBR®Green I (Molecular Probes). If more specific detection is required,fluorescence-based technologies can monitor the appearance of a specificproduct during the reaction. The use of dual-labeled fluorogenic probessuch as in the TaqMan™ system (Applied Biosystems) which utilizes the5′-3′ exonuclease activity of the Taq polymerase is a good example(Livak K. J. et al. 1995, PCR Methods Appl. 4:357-362). TaqMan™ can beperformed during amplification and this “real-time” detection can bedone in a single closed tube hence eliminating post-PCR sample handlingand consequently preventing the risk of amplicon carryover. Severalother fluorescence-based detection methods can be performed inreal-time. Fluorescence resonance energy transfer (FRET) is theprinciple behind the use of adjacent hybridization probes (Wittwer, C.T. et al. 1997. BioTechniques 22:130-138), molecular beacons (Tyagi S.and Kramer F. R. 1996. Nature Biotechnology 14:303-308) and scorpions(Whitcomb et al. 1999. Nature Biotechnology 17:804-807). Adjacenthybridization probes are designed to be internal to the amplificationprimers. The 3′ end of one probe is labelled with a donor fluorophorewhile the 5′ end of an adjacent probe is labelled with an acceptorfluorophore. When the two probes are specifically hybridized in closedproximity (spaced by 1 to 5 nucleotides) the donor fluorophore which hasbeen excited by an external light source emits light that is absorbed bya second acceptor that emit more fluorescence and yields a FRET signal.Molecular beacons possess a stem-and-loop structure where the loop isthe probe and at the bottom of the stem a fluorescent moiety is at oneend while a quenching moiety is at the other end. The beacons undergo afluorogenic conformational change when they hybridize to their targetshence separating the fluorochrome from its quencher. The FRET principleis also used in an air thermal cycler with a built-in fluorometer(Wittwer, C. T. et al. 1997. BioTechniques 22:130-138). Theamplification and detection are extremely rapid as reactions areperformed in capillaries: it takes only 18 min to complete 45 cycles.Those techniques are suitable especially in the case where few pathogensare searched for. Boehringer-Roche Inc. sells the LightCycler™, andCepheid makes the SmartCycler. These two apparatus are capable of rapidcycle PCR combined with fluorescent SYBR® Green I or FRET detection. Werecently demonstrated in our laboratory, real-time detection of 10 CFUin less than 40 minutes using adjacent hybridization probes on theLightCycler™. Methods based on the detection of fluorescence areparticularly promising for utilization in routine diagnosis as they arevery rapid, quantitative and can be automated.

Microbial pathogens detection and identification may also be performedby solid support or liquid hybridization using species-specific internalDNA probes hybridizing to an amplification product. Such probes may begenerated from any sequence from our repertory and designed tospecifically hybridize to DNA amplification products which are objectsof the present invention. Alternatively, the internal probes for speciesor genus or family or group detection and identification may be derivedfrom the amplicons produced by a universal, family-, group-, genus- orspecies-specific amplification assay(s). The oligonucleotide probes maybe labeled with biotin or with digoxigenin or with any other reportermolecule (for more details see below the section on hybrid capture).Hybrization on a solid support is amendable to miniaturization.

At present the oligonucleotide nucleic acid microarray technology isappealing. Currently, available low to medium density arrays (Heller etal., An integrated microelectronics hybridization system for genomicresearch and diagnostic applications. In: Harrison, D. J., and van denBerg, A., 1998, Micro total analysis systems '98, Kluwer AcademicPublisher, Dordrecht.) could specifically capture fluorescent-labelledamplicons. Detection methods for hybridization are not limited tofluorescence; potentiometry, colorimetry and plasmon resonance are someexamples of alternative detection methods. In addition to detection byhybridization, nucleic acid microarrays could be used to perform rapidsequencing by hybridization. Mass spectrometry could also be applicablefor rapid identification of the amplicon or even for sequencing of theamplification products (Chiu and Cantor, 1999, Clinical Chemistry45:1578; Berkenkamp et al., 1998, Science 281:260).

For the future of our assay format, we also consider the major challengeof molecular diagnostics tools, i.e.: integration of the major stepsincluding sample preparation, genetic amplification, detection, dataanalysis and presentation (Anderson et al., Advances in integratedgenetic analysis. In: Harrison, D. J., and van den Berg, A., 1998, Micrototal analysis systems '98, Kluwer Academic Publisher, Dordrecht.).

To ensure PCR efficiency, glycerol, dimethyl sulfoxide (DMSO) or otherrelated solvents can be used to increase the sensitivity of the PCR andto overcome problems associated with the amplification of a target DNAhaving a high GC content or forming strong secondary structures(Dieffenbach and Dveksler, 1995, PCR Primer: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Plainview, N.Y.). The concentrationranges for glycerol and DMSO are 5-15% (v/v) and 3-10% (v/v),respectively. For the PCR reaction mixture, the concentration ranges forthe amplification primers and MgCl₂ are 0.1-1.5 μM and 1.0-10.0 mM,respectively. Modifications of the standard PCR protocol using externaland nested primers (i.e. nested PCR) or using more than one primer pair(i.e. multiplex PCR) may also be used (Persing et al., 1993, DiagnosticMolecular Microbiology: Principles and Applications, American Societyfor Microbiology, Washington, D.C.). For more details about the PCRprotocols and amplicon detection methods, see Examples.

Hybrid Capture and Chemiluminescence Detection of Amplification Products

Hybridization and detection of amplicons by chemiluminescence wereadapted from Nikiforov et al. (1994, PCR Methods and Applications3:285-291 and 1995, Anal. Biochem. 227:201-209) and from the DIG™ systemprotocol of Boehringer Mannheim. Briefly, 50 μl of a 25 picomolessolution of capture probe diluted in EDC{1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride} areimmobilized in each well of 96-wells plates (Microlite™ 2, Dynex) byincubation overnight at room temperature. The next day, the plates areincubated with a solution of 1% BSA diluted into TNTw (10 mM Tris-HCl,pH 7.5; 150 mM NaCl; 0.05% Tween™ 20) for 1 hour at 37° C. The platesare then washed on a Wellwash Ascent™ (Labsystems) with TNTw followed byWashing Buffer (100 mM maleic acid pH7.5; 150 mM NaCl; 0.3% Tween™ 20).

The amplicons were labelled with DIG-11-dUTP during PCR using the PCRDIG Labelling Mix from Boehringer Mannheim according to themanufacturer's instructions. Hybridization of the amplicons to thecapture probes is performed in triplicate at stringent temperature(generally, probes are designed to allow hybrization at 55° C., thestringent temperature) for 30 minutes in 1.5 M NaCl; 10 mM EDTA. It isfollowed by two washes in 2×SSC; 0.1% SDS, then by four washes in0.1×SSC; 0.1% SDS at the stringent temperature (55° C.). Detection with1,2 dioxetane chemiluminescent alkaline phosphatase substrates likeCSPD® (Tropix Inc.) is performed according to the manufacturer'sinstructions but with shorter incubations times and a different antibodyconcentration. The plates are agitated at each step, the blockingincubation is performed for only 5 minutes, the anti-DIG-AP1 is used ata 1:1000 dilution, the incubation with antibody lasts 15 minutes, theplates are washed twice for only 5 minutes. Finally, after a 2 minutesincubation into the detection buffer, the plates are incubated 5 minuteswith CSPD® at room temperature followed by a 10 minutes incubation at37° C. without agitation. Luminous signal detection is performed on aDynex Microtiter Plate Luminometer using RLU (Relative Light Units).

Specificity, Ubiquity and Sensitivity Tests for Oligonucleotide Primersand Probes

The specificity of oligonucleotide primers and probes was tested byamplification of DNA or by hybridization with bacterial or fungal orparasitical species selected from a panel comprising closely relatedspecies and species sharing the same anatomo-pathological site (seeTables and Examples). All of the bacterial, fungal and parasiticalspecies tested were likely to be pathogens associated with infections orpotential contaminants which can be isolated from clinical specimens.Each target DNA could be released from microbial cells using standardchemical and/or physical treatments to lyse the cells (Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.) or alternatively,genomic DNA purified with the GNOME™ DNA kit (Bio101, Vista, Calif.) wasused. Subsequently, the DNA was subjected to amplification with theprimer pairs. Specific primers or probes amplified only the targetmicrobial species, genus, family or group.

Oligonucleotides primers found to amplify specifically the targetspecies, genus, family or group were subsequently tested for theirubiquity by amplification (i.e. ubiquitous primers amplified efficientlymost or all isolates of the target species or genus or family or group).Finally, the sensitivity of the primers or probes was determined byusing 10-fold or 2-fold dilutions of purified genomic DNA from thetargeted microorganism. For most assays, sensitivity levels in the rangeof 1-100 copies were obtained. The specificity, ubiquity and sensitivityof the PCR assays using the selected amplification primer pairs weretested either directly from cultures of microbial species or frompurified microbial genomic DNA.

Probes were tested in hybrid capture assays as described above. Anoligonucleotide probe was considered specific only when it hybridizedsolely to DNA from the species or genus or family or group from which itwas selected. Oligonucleotide probes found to be specific weresubsequently tested for their ubiquity (i.e. ubiquitous probes detectedefficiently most or all isolates of the target species or genus orfamily or group) by hybridization to microbial DNAs from differentclinical isolates of the species or genus or family or group of interestincluding ATCC reference strains. Similarly, oligonucleotide primers andprobes could be derived from antimicrobial agents resistance or toxingenes which are objects of the present invention.

Reference Strains

The reference strains used to build proprietary tuf, atpD and recAsequence data subrepertories, as well as to test the amplification andhybridization assays were obtained from (i) the American Type CultureCollection (ATCC), (ii) the Laboratoire de santé publique du Québec(LSPQ), (iii) the Centers for Disease Control and Prevention (CDC), (iv)the National Culture Type Collection (NCTC) and (v) several otherreference laboratories throughout the world. The identity of ourreference strains was confirmed by phenotypic testing and reconfirmed byanalysis of tuf, atpD and recA sequences (see Example 13).

Antimicrobial Agents Resistance Genes

Antimicrobial resistance complicates treatment and often leads totherapeutic failures. Furthermore, overuse of antibiotics inevitablyleads to the emergence of microbial resistance. Our goal is to provideclinicians, in approximately one hour, the needed information toprescribe optimal treatments. Besides the rapid identification ofnegative clinical specimens with DNA-based tests for universal algal,archaeal, bacterial, fungal or parasitical detection and theidentification of the presence of a specific pathogen in the positivespecimens with species- and/or genus- and/or family- and/orgroup-specific DNA-based tests, clinicians also need timely informationabout the ability of the microbial pathogen to resist antibiotictreatments. We feel that the most efficient strategy to evaluate rapidlymicrobial resistance to antimicrobials is to detect directly from theclinical specimens the most common and clinically importantantimicrobial agents resistance genes (i.e. DNA-based tests for thespecific detection of antimicrobial agents resistance genes). Since thesequence from the most important and common antimicrobial agentsresistance genes are available from public databases, our strategy is touse the sequence from a portion or from the entire resistance gene todesign specific oligonucleotide primers or probes which will be used asa basis for the development of sensitive and rapid DNA-based tests. Thelist of each of the antimicrobial agents resistance genes selected onthe basis of their clinical relevance (i.e. high incidence andimportance) is given in Table 5; descriptions of the designedamplification primers and internal probes are given in Tables 72-75, 77,83, and 88-89. Our approach is unique because the antimicrobial agentsresistance genes detection and the microbial detection andidentification can be performed simultaneously, or independently, orsequentially in multiplex or parallel or sequential assays under uniformPCR amplification conditions. These amplifications can also be doneseparately.

Toxin Genes

Toxin identification is often very important to prescribe optimaltreatments. Besides the rapid identification of negative clinicalspecimens with DNA-based tests for universal bacterial detection and theidentification of the presence of a specific pathogen in the positivespecimens with species- and/or genus- and/or family- and/orgroup-specific DNA-based tests, clinicians sometimes need timelyinformation about the ability of certain bacterial pathogens to producetoxins. Since the sequence from the most important and common bacterialtoxin genes are available from public databases, our strategy is to usethe sequence from a portion or from the entire toxin gene to designspecific oligonucleotide primers or probes which will be used as a basisfor the development of sensitive and rapid DNA-based tests. The list ofeach of the bacterial toxin genes selected on the basis of theirclinical relevance (i.e. high incidence and importance) is given inTable 6; descriptions of the designed amplification primers and internalprobes are given in Tables 60, 70 and 71. Our approach is unique becausethe toxin genes detection and the bacterial detection and identificationcan be performed simultaneously, or independently, or sequentially, inmultiplex or parallel or sequential assays under uniform PCRamplification conditions. These amplifications can also be doneseparately.

Universal Bacterial Detection

In the routine microbiology laboratory, a high percentage of clinicalspecimens sent for bacterial identification are negative by culture.Testing clinical samples with universal amplification primers oruniversal probes to detect the presence of bacteria prior to specificidentification and screening out the numerous negative specimens is thususeful as it reduces costs and may rapidly orient the clinicalmanagement of the patients. Several amplification primers and probeswere therefore synthesized from highly conserved portions of bacterialsequences from the tuf, atpD and recA nucleic acids and/or sequences.The universal primers selection was based on a multiple sequencealignment constructed with sequences from our repertory.

All computer analysis of amino acid and nucleotide sequences wereperformed by using the GCG programs. Subsequently, optimal PCR primersfor the universal amplification of bacteria were selected with the helpof the Oligo™ program. The selected primers are degenerated at severalnucleotide positions and contain several inosines in order to allow theamplification of all clinically relevant bacterial species. Inosine is anucleotide analog able to specifically bind to any of the fournucleotides A, C, G or T. Degenerated oligonucleotides consist of anoligonucleotide mix having two or more of the four nucleotides A, C, Gor T at the site of mismatches. The inclusion of inosine and/or of baseambiguities in the amplification primers allow mismatch tolerancethereby permitting the amplification of a wider array of targetnucleotide sequences (Dieffenbach and Dveksler, 1995 PCR Primer: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Plainview,N.Y.).

The amplification conditions with the universal primers are very similarto those used for the species- and genus-specific amplification assaysexcept that the annealing temperature is slightly lower. The originaluniversal PCR assay described in our assigned WO98/20157 (SEQ ID NOs.23-24 of the latter application) was specific and nearly ubiquitous forthe detection of bacteria. The specificity for bacteria was verified byamplifying genomic DNA isolated from the 12 fungal species as well asgenomic DNA from Leishmania donovani, Saccharomyces cerevisiae and humanlymphocytes. None of the above eukaryotic DNA preparations could beamplified by the universal assay, thereby suggesting that this test isspecific for bacteria. The ubiquity of the universal assay was verifiedby amplifying genomic DNAs from 116 reference strains which represent 95of the most clinically relevant bacterial species. These species havebeen selected from the bacterial species listed in Table 4. We foundthat at least 104 of these strains could be amplified. However, theassay could be improved since bacterial species which could not beamplified with the original tuf nucleic acids and/or sequences-basedassay included species belonging to the following genera:Corynebacterium (11 species) and Stenotrophomonas (1 species).Sequencing of the tuf genes from these bacterial species and others hasbeen performed in the scope of the present invention in order to improvethe universal assay. This sequencing data has been used to select newuniversal primers which may be more ubiquitous and more sensitive. Also,we improved our primer and probes design strategy by taking intoconsideration the phylogeny observed in analysing our repertory of tuf,atpD and recA sequences. Data from each of the 3 main subrepertories(tuf, atpD and recA) was subjected to a basic phylogenic analysis usingthe Pileup command from version 10 of the GCG package (Genetics ComputerGroup, inc.). This analysis indicated the main branches or phylareflecting the relationships between sequences. Instead of trying todesign primers or probes able to hybridize to all phyla, we designedprimers or probes able to hybridize to the main phyla while trying touse the largest phylum possible. This strategy should allow lessdegenerated primers hence improving sensitivity and by combining primersin a mutiplex assay, improve ubiquity. Universal primers SEQ ID NOs.643-645 based on tuf sequences have been designed to amplify mostpathogenic bacteria except Actinomyceteae, Clostridiaceae and theCytophaga, Flexibacter and Bacteroides phylum (pathogenic bacteria ofthis phylum include mostly Bacteroides, Porphyromonas and Prevotellaspecies). Primers to fill these gaps have been designed forActinomyceteae (SEQ ID NOs. 646-648), Clostridiaceae (SEQ ID NOs.796-797, 808-811), and the Cytophaga, Flexibacter and Bacteroides phylum(SEQ ID NOs. 649-651), also derived from tuf nucleic acids and/orsequences. These primers sets could be used alone or in conjuction torender the universal assay more ubiquitous.

Universal primers derived from atpD sequences include SEQ ID NOs.562-565. Combination of these primers does not amplify human DNA butshould amplify almost all pathogenic bacterial species exceptproteobacteria belonging to the epsilon subdivision (Campylobacter andHelicobacter), the bacteria from the Cytophaga, Flexibacter andBacteroides group and some actinomycetes and corynebacteria. Byanalysing atpD sequences from the latter species, primers and probes tospecifically fill these gaps could be designed and used in conjuctionwith primers SEQ ID NOs. 562-565, also derived from atpD nucleic acidsand/or sequences.

In addition, universality of the assay could be expanded by mixing atpDsequences-derived primers with tuf sequences-derived primers.Ultimately, even recA sequences-derived primers could be added to fillsome gaps in the universal assay.

It is important to note that the 95 bacterial species selected to testthe ubiquity of the universal assay include all of the most clinicallyrelevant bacterial species associated with a variety of human infectionsacquired in the community or in hospitals (nosocomial infections). Themost clinically important bacterial and fungal pathogens are listed inTables 1 and 2.

Amino Acid Sequences Derived from tuf, atpD and recA Nucleic Acidsand/or Sequences

The amino acid sequences translated from the repertory of tuf, atpD andrecA nucleic acids and/or sequences are also an object of the presentinvention. The amino acid sequence data will be particularly useful forhomology modeling of three-dimensional (3D) structure of the elongationfactor Tu, elongation factor G, elongation factor 1a, ATPase subunitbeta and RecA recombinase. For all these proteins, at least onestructure model has been published using X-ray diffraction data fromcrystals. Based on those structural informations it is possible to usecomputer sofware to build 3D model structures for any other proteinhaving peptide sequence homologies with the known structure (Greer,1991, Methods in Enzymology, 202:239-252; Taylor, 1994, TrendsBiotechnol., 12(5):154-158; Sali, 1995, Curr. Opin. Biotechnol.6:437-451; Sanchez and Sali, 1997, Curr. Opin. Struct. Biol. 7:206-214;Fischer and Eisenberg, 1999, Curr. Opin. Struct. Biol. 9:208-211; Guexet al., 1999, Trends Biochem. Sci. 24: 364-367). Model structures oftarget proteins are used for the design or to predict the behavior ofligands and inhibitors such as antibiotics. Since EF-Tu and EF-G arealready known as antibiotic targets (see above) and since the betasubunit of ATPase and RecA recombinase are essential to the survival ofthe microbial cells in natural conditions of infection, all fourproteins could be considered antibiotic targets. Sequence data,especially the new data generated by us could be very useful to assistthe creation of new antibiotic molecules with desired spectrum ofactivity. In addition, model structures could be used to improve proteinfunction for commercial purposes such as improving antibiotic productionby microbial strains or increasing biomass.

The following detailed embodiments and appended drawings are provided asillustrative examples of his invention, with no intention to limit thescope thereof.

EXAMPLES AND TABLES

For sake of clarity, here is a list of Examples and Tables:

Example 1: Sequencing of bacterial atpD (F-type and V-type) genefragments.

Example 2: Sequencing of eukaryotic atpD (F-type and V-type) genefragments.

Example 3: Sequencing of eukaryotic tuf (EF-1) gene fragments.

Example 4: Sequencing of eukaryotic tuf (organelle origin, M) genefragments.

Example 5: Specific detection and identification of Streptococcusagalactiae using tuf sequences.

Example 6: Specific detection and identification of Streptococcusagalactiae using atpD sequences.

Example 7: Development of a PCR assay for detection and identificationof staphylococci at genus and species levels.

Example 8: Differentiating between the two closely related yeast speciesCandida albicans and Candida dubliniensis.

Example 9: Specific detection and identification of Entamoebahistolytica.

Example 10: Sensitive detection and identification of Chlamydiatrachomatis.

Example 11: Genus-specific detection and identification of enterococci.

Example 12: Detection and identification of the major bacterialplatelets contaminants using tuf sequences with a multiplex PCR test.

Example 13: The resolving power of the tuf and atpD sequences databasesis comparable to the biochemical methods for bacterial identification.

Example 14: Detection of group B streptococci from clinical specimens.

Example 15: Simultaneous detection and identification of Streptococcuspyogenes and its pyrogenic exotoxin A.

Example 16: Real-time detection and identification of Shigatoxin-producing bacteria.

Example 17: Development of a PCR assay for the detection andidentification of staphylococci at genus and species levels and itsassociated mecA gene.

Example 18: Sequencing of pbp1a, pbp2b and pbp2x genes of Streptoccoccuspneumoniae.

Example 19: Sequencing of hexA genes of Streptococcus species.

Example 20: Development of a multiplex PCR assay for the detection ofStreptococcus pneumoniae and its penicillin resistance genes.

Example 21: Sequencing of the vancomycin resistance vanA, vanC1, vanC2and vanC3 genes.

Example 22: Development of a PCR assay for the detection andidentification of enterococci at genus and species levels and itsassociated resistance genes vanA and vanB.

Example 23: Development of a multiplex PCR assay for detection andidentification of vancomycin-resistant Enterococcus faecalis,Enterococcus faecium, Enterococcus gallinarum, Enterococcuscasseliflavus, and Enterococcus flavescens.

Example 24: Universal amplification involving the EF-G (fusA)subdivision of tuf sequences.

Example 25: DNA fragment isolation from Staphylococcus saprophyticus byarbitrarily primed PCR.

Example 26: Sequencing of prokaryotic tuf gene fragments.

Example 27: Sequencing of procaryotic recA gene fragments.

Example 28: Specific detection and identification of Escherichiacoli/Shigella sp. using tuf sequences.

Example 29: Specific detection and identification of Klebsiellapneumoniae using atpD sequences.

Example 30: Specific detection and identification of Acinetobacterbaumanii using tuf sequences.

Example 31: Specific detection and identification of Neisseriagonorrhoeae using tuf sequences.

Example 32: Sequencing of bacterial gyrA and parC gene fragments.

Example 33: Development of a PCR assay for the specific detection andidentification of Staphylococcus aureus and its quinolone resistancegenes gyrA and parC.

Example 34: Development of a PCR assay for the detection andidentification of Klebsiella pneumoniae and its quinolone resistancegenes gyrA and parC.

Example 35: Development of a PCR assay for the detection andidentification of Streptococcus pneumoniae and its quinolone resistancegenes gyrA and parC.

Example 36: Detection of extended-spectrum TEM-type β-lactamases inEscherichia coli.

Example 37: Detection of extended-spectrum SHV-type β-lactamases inKlebsiella pneumoniae.

Example 38: Development of a PCR assay for the detection andidentification of Neisseria gonorrhoeae and its associated tetracyclineresistance gene tetM.

Example 39: Development of a PCR assay for the detection andidentification of Shigella sp. and their associated trimethoprimresistance gene dhfr1a.

Example 40: Development of a PCR assay for the detection andidentification of Acinetobacter baumanii and its associatedaminoglycoside resistance gene aph(3)-VIa.

Example 41: Specific detection and identification of Bacteroidesfragilis using atpD (V-type) sequences.

Example 42: Evidence for horizontal gene transfer in the evolution ofthe elongation factor Tu in Enterococci.

Example 43: Elongation factor Tu (tuf) and the F-ATPase beta-subunit(atpD) as phylogenetic tools for species of the familyEnterobacteriaceae.

Example 44: Testing new pairs of PCR primers selected from twospecies-specific genomic DNA fragments which are objects of U.S. Pat.No. 6,001,564.

Example 45: Testing modified versions of PCR primers derived from thesequence of several primers which are objects of U.S. Pat. No.6,001,564.

The various Tables show the strategies used for the selection of avariety of DNA amplification primers, nucleic acid hybridization probesand molecular beacon internal probes:

-   -   (i) Table 39 shows the amplification primers used for nucleic        acid amplification from tuf sequences.    -   (ii) Table 40 shows the amplification primers used for nucleic        acid amplification from atpD sequences.    -   (iii) Table 41 shows the internal hybridization probes for        detection of tuf sequences.    -   (iv) Table 42 illustrates the strategy used for the selection of        the amplification primers specific for atpD sequences of the        F-type.    -   (v) Table 43 illustrates the strategy used for the selection of        the amplification primers specific for atpD sequences of the        V-type.    -   (vi) Table 44 illustrates the strategy used for the selection of        the amplification primers specific for the tuf sequences of        organelle lineage (M, the letter M is used to indicate that in        most cases, the organelle is the mitochondria).    -   (vii) Table 45 illustrates the strategy used for the selection        of the amplification primers specific for the tuf sequences of        eukaryotes (EF-1).    -   (viii) Table 46 illustrates the strategy for the selection of        Streptococcus agalactiae-specific amplification primers from tuf        sequences.    -   (ix) Table 47 illustrates the strategy for the selection of        Streptococcus agalactiae-specific hybridization probes from tuf        sequences.    -   (x) Table 48 illustrates the strategy for the selection of        Streptococcus agalactiae-specific amplification primers from        atpD sequences.    -   (xi) Table 49 illustrates the strategy for the selection from        tuf sequences of Candida albicans/dubliniensis-specific        amplification primers, Candida albicans-specific hybridization        probe and Candida dubliniensis-specific hybridization probe.    -   (xii) Table 50 illustrates the strategy for the selection of        Staphylococcus-specific amplification primers from tuf        sequences.    -   (xiii) Table 51 illustrates the strategy for the selection of        the Staphylococcus-specific hybridization probe from tuf        sequences.    -   (xiv) Table 52 illustrates the strategy for the selection of        Staphylococcus saprophyticus-specific and Staphylococcus        haemolyticus-specific hybridization probes from tuf sequences.    -   (xv) Table 53 illustrates the strategy for the selection of        Staphylococcus aureus-specific and Staphylococcus        epidermidis-specific hybridization probes from tuf sequences.    -   (xvi) Table 54 illustrates the strategy for the selection of the        Staphylococcus hominis-specific hybridization probe from tuf        sequences.    -   (xvii) Table 55 illustrates the strategy for the selection of        the Enterococcus-specific amplification primers from tuf        sequences.    -   (xviii) Table 56 illustrates the strategy for the selection of        the Enterococcus faecalis-specific hybridization probe, of the        Enterococcus faecium-specific hybridization probe and of the        Enterococcus casseliflavus-flavescens-gallinarum group-specific        hybridization probe from tuf sequences.    -   (xix) Table 57 illustrates the strategy for the selection of        primers from tuf sequences for the identification of platelets        contaminants.    -   (xx) Table 58 illustrates the strategy for the selection of the        universal amplification primers from atpD sequences.    -   (xxi) Table 59 shows the amplification primers used for nucleic        acid amplification from recA sequences.    -   (xxii) Table 60 shows the specific and ubiquitous primers for        nucleic acid amplification from speA sequences.    -   (xxiii) Table 61 illustrates the first strategy for the        selection of Streptococcus pyogenes-specific amplification        primers from speA sequences.    -   (xxiv) Table 62 illustrates the second strategy for the        selection of Streptococcus pyogenes-specific amplification        primers from speA sequences.    -   (xxv) Table 63 illustrates the strategy for the selection of        Streptococcus pyogenes-specific amplification primers from tuf        sequences.    -   (xxvi) Table 64 illustrates the strategy for the selection of        stc₁-specific amplification primers and hybridization probe.    -   (xxxii) Table 65 illustrates the strategy for the selection of        stc₂-specific amplification primers and hybridization probe.    -   (xxviii) Table 66 illustrates the strategy for the selection of        vanA-specific amplification primers from van sequences.    -   (xxix) Table 67 illustrates the strategy for the selection of        vanB-specific amplification primers from van sequences.    -   (xxx) Table 68 illustrates the strategy for the selection of        vanC-specific amplification primers from vanC sequences.    -   (xxxi) Table 69 illustrates the strategy for the selection of        Streptococcus pneumoniae-specific amplification primers and        hybridization probes from pbp1a sequences.    -   (xxxii) Table 70 shows the specific and ubiquitous primers for        nucleic acid amplification from toxin gene sequences.    -   (xxxiii) Table 71 shows the molecular beacon internal        hybridization probes for specific detection of toxin sequences.    -   (xxxiv) Table 72 shows the specific and ubiquitous primers for        nucleic acid amplification from van sequences.    -   (xxxv) Table 73 shows the internal hybridization probes for        specific detection of van sequences.    -   (xxxvi) Table 74 shows the specific and ubiquitous primers for        nucleic acid amplification from pbp sequences.    -   (xxxvii) Table 75 shows the internal hybridization probes for        specific detection of pbp sequences.    -   (xxxviii) Table 76 illustrates the strategy for the selection of        vanAB-specific amplification primers and vanA- and vanB-specific        hybridization probes from van sequences.    -   (xxxix) Table 77 shows the internal hybridization probe for        specific detection of mecA.    -   (xl) Table 78 shows the specific and ubiquitous primers for        nucleic acid amplification from hexA sequences.    -   (xli) Table 79 shows the internal hybridization probe for        specific detection of hexA.    -   (xlii) Table 80 illustrates the strategy for the selection of        Streptococcus pneumoniae species-specific amplification primers        and hybridization probe from hexA sequences.    -   (xliii) Table 81 shows the specific and ubiquitous primers for        nucleic acid amplification from pcp sequences.    -   (xliv) Table 82 shows specific and ubiquitous primers for        nucleic acid amplification of S. saprophyticus sequences of        unknown coding potential.    -   (xlv) Table 83 shows the molecular beacon internal hybridization        probes for specific detection of antimicrobial agents resistance        gene sequences.    -   (xlvi) Table 84 shows the molecular beacon internal        hybridization probe for specific detection of S. aureus gene        sequences of unknown coding potential.    -   (xlvii) Table 85 shows the molecular beacon hybridization        internal probe for specific detection of tuf sequences.    -   (xlviii) Table 86 shows the molecular beacon internal        hybridization probes for specific detection of ddl and mtl        sequences.    -   (xlix) Table 87 shows the internal hybridization probe for        specific detection of S. aureus sequences of unknown coding        potential.    -   (l) Table 88 shows the amplification primers used for nucleic        acid amplification from antimicrobial agents resistance genes        sequences.    -   (li) Table 89 shows the internal hybridization probes for        specific detection of antimicrobial agents resistance genes        sequences.    -   (lii) Table 90 shows the molecular beacon internal hybridization        probes for specific detection of atpD sequences.    -   (liii) Table 91 shows the internal hybridization probes for        specific detection of atpD sequences.    -   (liv) Table 92 shows the internal hybridization probes for        specific detection of ddl and mtl sequences.

As shown in these Tables, the selected amplification primers may containinosines and/or base ambiguities. Inosine is a nucleotide analog able tospecifically bind to any of the four nucleotides A, C, G or T.Alternatively, degenerated oligonucleotides which consist of anoligonucleotide mix having two or more of the four nucleotides A, C, Gor T at the site of mismatches were used. The inclusion of inosineand/or of degeneracies in the amplification primers allows mismatchtolerance thereby permitting the amplification of a wider array oftarget nucleotide sequences (Dieffenbach and Dveksler, 1995 PCR Primer:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview,N.Y.).

EXAMPLES Example 1

Sequencing of bacterial atpD (F-type and V-type) gene fragments.

As shown in Table 42, the comparison of publicly available atpD (F-type)sequences from a variety of bacterial species revealed conserved regionsallowing the design of PCR primers able to amplify atpD sequences(F-type) from a wide range of bacterial species. Using primers pairs SEQID NOs. 566 and 567, 566 and 814, 568 and 567, 570 and 567, 572 and 567,569 and 567, 571 and 567, 700 and 567, it was possible to amplify andsequence atpD sequences SEQ ID NOs. 242-270, 272-398, 673-674, 737-767,866-867, 942-955, 1245-1254, 1256-1265, 1527, 1576, 1577, 1600-1604,1640-1646, 1649, 1652, 1655, 1657, 1659-1660, 1671, 1844-1845, and1849-1865.

Similarly, Table 43 shows the strategy to design the PCR primers able toamplify atpD sequences of the V-type from a wide range of archaeal andbacterial species. Using primers SEQ ID NOs. 681-683, it was possible toamplify and sequence atpD sequences SEQ ID NOs. 827-832, 929-931, 958and 966. As the gene was difficult to amplify for several species,additional amplification primers were designed inside the originalamplicon (SEQ ID NOs. 1203-1207) in order to obtain sequence informationfor these species. Other primers (SEQ ID NO. 1212, 1213, 2282-2285) werealso designed to amplify regions of the atpD gene (V-type) inarchaebacteria.

Example 2

Sequencing of Eukaryotic atpD (F-Type and V-Type) Gene Fragments.

The comparison of publicly available atpD (F-type) sequences from avariety of fungal and parasitical species revealed conserved regionsallowing the design of PCR primers able to amplify atpD sequences from awide range of fungal and parasitical species. Using primers pairs SEQ IDNOs. 568 and 573, 574 and 573, 574 and 708, and 566 and 567, it waspossible to amplify and sequence atpD sequences SEQ ID NOs. 458-497,530-538, 663, 667, 676, 678-680, 768-778, 856-862, 889-896, 941,1638-1639, 1647, 1650-1651, 1653-1654, 1656, 1658, 1684, 1846-1848, and2189-2192.

In the same manner, the primers described in Table 43 (SEQ ID NOs.681-683) could amplify the atpD (V-type) gene from various fungal andparasitical species. This strategy allowed to obtain SEQ ID NOs.834-839, 956-957, and 959-965.

Example 3

Sequencing of Eukaryotic tuf (EF-1) Gene Fragments.

As shown in Table 45, the comparison of publicly available tuf (EF-1)sequences from a variety of fungal and parasitical species revealedconserved regions allowing the design of PCR primers able to amplify tufsequences from a wide range of fungal and parasitical species. Usingprimers pairs SEQ ID NOs. 558 and 559, 813 and 559, 558 and 815, 560 and559, 653 and 559, 558 and 655, and 654 and 559, 1999 and 2000, 2001 and2003, 2002 and 2003, it was possible to amplify and sequence tufsequences SEQ ID NOs. 399-457, 509-529, 622-624, 677, 779-790, 840-842,865, 897-903, 1266-1287, 1561-1571 and 1685.

Example 4

Sequencing of Eukaryotic tuf (Organelle Origin, M) Gene Fragments.

As shown in Table 44, the comparison of publicly available tuf(organelle origin, M) sequences from a variety of fungal and parasiticalorganelles revealed conserved regions allowing the design of PCR primersable to amplify tuf sequences of several organelles belonging to a widerange fungal and parasitical species. Using primers pairs SEQ ID NOs.664 and 652, 664 and 561, 911 and 914, 912 and 914, 913 and 915, 916 and561, 664 and 917, it was possible to amplify and sequence tuf sequencesSEQ ID NOs. 498-508, 791-792, 843-855, 904-910, 1664, 1666-1667,1669-1670, 1673-1683, 1686-1689, 1874-1876, 1879, 1956-1960, and2193-2199.

Example 5

Specific Detection and Identification of Streptococcus agalactiae Usingtuf Sequences.

As shown in Table 46, the comparison of tuf sequences from a variety ofbacterial species allowed the selection of PCR primers specific for S.agalactiae. The strategy used to design the PCR primers was based on theanalysis of a multiple sequence alignment of various tuf sequences. Themultiple sequence alignment includes the tuf sequences of four bacterialstrains from the target species as well as tuf sequences from otherspecies and bacterial genera, especially representatives of closelyrelated species. A careful analysis of this alignment allowed theselection of oligonucleotide sequences which are conserved within thetarget species but which discriminate sequences from other species andgenera, especially from the closely related species, thereby permittingthe species-specific, ubiquitous and sensitive detection andidentification of the target bacterial species.

The chosen primer pair, oligos SEQ ID NO. 549 and SEQ ID NO. 550, givesan amplification product of 252 bp. Standard PCR was carried out using0.4 μM of each primer, 2.5 mM MgCl₂, BSA 0.05 mM, 1×Taq Buffer(Promega), dNTP 0.2 mM (Pharmacia), 0.5 U Taq DNA polymerase (Promega)coupled with TaqStart™ antibody (Clontech Laboratories Inc., Palo Alto),1 μl of genomic DNA sample in a final volume of 20 μl using a PTC-200thermocycler (MJ Research Inc.). The optimal cycling conditions formaximum sensitivity and specificity were 3 minutes at 95° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 62° C., followed by terminal extension at 72°C. for 2 minutes. Detection of the PCR products was made byelectrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidiumbromide.

Specificity of the assay was tested by adding into the PCR reactions,0.1 ng of genomic DNA from each of the bacterial species listed in Table8. Efficient amplification was observed only for the 5 S. agalactiaestrains listed. Of the other bacterial species, including 32 speciesrepresentative of the vaginal flora and 27 other streptococcal species,only S. acidominimus yielded amplification. The signal with 0.1 ng of S.acidominimus genomic DNA was weak and the detection limit for thisspecies was 10 pg (corresponding to more than 4000 genome copies) whilethe detection limit for S. agalactiae was 2.5 fg (corresponding to onegenome copy) of genomic DNA.

To increase the specificity of the assay, internal probes were designedfor FRET (Fluorescence Resonance Energy Transfer) detection using theLightCycler™ (Idaho Technology). As illustrated in Table 47, a multiplesequence alignment of streptococcal tuf sequence fragments correspondingto the 252 bp region amplified by primers SEQ ID NO. 549 and SEQ ID NO.550, was used for the design of internal probes TSagHF436 (SEQ ID NO.582) and TSagHF465 (SEQ ID NO. 583). The region of the amplicon selectedfor internal probes contained sequences unique and specific to S.agalactiae. SEQ ID NO. 583, the more specific probe, is labelled withfluorescein in 3′, while SEQ ID NO. 582, the less discriminant probe, islabelled with CY5 in 5′ and blocked in 3′ with a phosphate group.However, since the FRET signal is only emitted if both probes areadjacently hybridized on the same target amplicon, detection is highlyspecific.

Real-time detection of PCR products using the LightCycler™ was carriedout using 0.4 μM of each primer (SEQ ID NO. 549-550), 0.2 μM of eachprobe (SEQ ID NO. 582-583), 2.5 mM MgCl₂, BSA 450 μg/ml, 1×PC2 Buffer(AB Peptides, St-Louis, Mo.), dNTP 0.2 mM (Pharmacia), 0.5 U KlenTaq1™DNA polymerase (AB Peptides) coupled with TaqStart™ antibody (ClontechLaboratories Inc., Palo Alto), 0.7 μl of genomic DNA sample in a finalvolume of 7 μl using a LightCycler thermocycler (Idaho Technology). Theoptimal cycling conditions for maximum sensitivity and specificity were3 minutes at 94° C. for initial denaturation, then forty cycles of threesteps consisting of 0 second (this setting meaning the LightCycler willreach the target temperature and stay at it for its minimal amount oftime) at 94° C., 10 seconds at 64° C., 20 seconds at 72° C.Amplification was monitored during each annealing steps using thefluorescence ratio. The streptococcal species having close sequencehomologies with the tuf sequence of S. agalactiae (S. acidominimus, S.anginosus, S. bovis, S. dysgalactiae, S. equi, S. ferus, S. gordonii, S.intermedius, S. parasanguis, S. parauberis, S. salivarius, S. sanguis,S. suis) as well as S. agalactiae were tested in the LightCycler with0.07 ng of genomic DNA per reaction. Only S. agalactiae yielded anamplification signal, hence demonstrating that the assay isspecies-specific. With the LightCycler™ assay using the internal FRETprobes, the detection limit for S. agalactiae was 1-2 genome copies ofgenomic DNA.

Example 6

Specific Detection and Identification of Streptococcus agalactiae UsingatpD Sequences.

As shown in Table 48, the comparison of atpD sequences from a variety ofbacterial species allowed the selection of PCR primers specific for S.agalactiae. The primer design strategy is similar to the strategydescribed in the preceding Example except that atpD sequences were usedin the alignment.

Four primers were selected, ASag42 (SEQ ID NO. 627), ASag52 (SEQ ID NO.628), ASag206 (SEQ ID NO. 625) and ASag371 (SEQ ID NO. 626). Thefollowing combinations of these four primers give four amplicons; SEQ IDNO. 627+SEQ ID NO. 625=190 bp, SEQ ID NO. 628+SEQ ID NO. 625=180 bp, SEQID NO. 627+SEQ ID NO. 626=355 bp, and SEQ ID NO. 628+SEQ ID NO. 626=345bp.

Standard PCR was carried out on PTC-200 thermocyclers (MJ Research Inc)using 0.4 μM of each primers pair, 2.5 mM MgCl₂, BSA 0.05 mM, 1×taqBuffer (Promega), dNTP 0.2 mM (Pharmacia), 0.5 U Taq DNA polymerase(Promega) coupled with TaqStart™ antibody (Clontech Laboratories Inc.,Palo Alto), 1 μl of genomic DNA sample in a final volume of 20 μL. Theoptimal cycling conditions for maximum sensitivity and specificity wereadjusted for each primer pair. Three minutes at 95° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at the optimal annealing temperature specifiedbelow were followed by terminal extension at 72° C. for 2 minutes.Detection of the PCR products was made by electrophoresis in agarosegels (2%) containing 0.25 μg/ml of ethidium bromide. Since atpDsequences are relatively more specific than tuf sequences, only the mostclosely related species namely, the steptococcal species listed in Table9, were tested.

All four primer pairs only amplified the six S. agalactiae strains. Withan annealing temperature of 63° C., the primer pair SEQ ID NO. 627+SEQID NO. 625 had a sensitivity of 1-5 fg (equivalent to 1-2 genomecopies). At 55° C., the primer pair SEQ ID NO. 628+SEQ ID NO. 625 had asensitivity of 2.5 fg (equivalent to 1 genome copy). At 60° C., theprimer pair SEQ ID NO. 627+SEQ ID NO. 626 had a sensitivity of 10 fg(equivalent to 4 genome copies). At 58° C., the primer pair SEQ ID NO.628+SEQ ID NO. 626 had a sensitivity of 2.5-5 fg (equivalent to 1-2genome copies). This proves that all four primer pairs can detect S.agalactiae with high specificity and sensitivity. Together with Example5, this example demonstrates that both tuf and atpD sequences aresuitable and flexible targets for the identification of microorganismsat the species level. The fact that 4 different primer pairs based onatpD sequences led to efficient and specific amplification of S.agalactiae demonstrates that the challenge is to find target genessuitable for diagnostic purposes, rather than finding primer pairs fromthese target sequences.

Example 7

Development of a PCR Assay for Detection and Identification ofStaphylococci at Genus and Species Levels.

Materials and Methods

Bacterial Strains.

The specificity of the PCR assay was verified by using a panel of ATCC(America Type Culture Collection) and DSMZ (Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH; German Collection ofMicroorganisms and Cell Cultures) reference strains consisting of 33gram-negative and 47 gram-positive bacterial species (Table 12). Inaddition, 295 clinical isolates representing 11 different species ofstaphylococci from the microbiology laboratory of the Centre HospitalierUniversitaire de Québec, Pavillon Centre Hospitalier de l'UniversitéLaval (CHUL) (Ste-Foy, Québec, Canada) were also tested to furthervalidate the Staphylococcus-specific PCR assay. These strains were allidentified by using (i) conventional methods or (ii) the automatedMicroScan Autoscan-4 system equipped with the Positive BP Combo PanelType 6 (Dade Diagnostics, Mississauga, Ontario, Canada). Bacterialstrains from frozen stocks kept at −80° C. in brain heart infusion (BHI)broth containing 10% glycerol were cultured on sheep blood agar or inBHI broth (Quelab Laboratories Inc, Montreal, Québec, Canada).

PCR Primers and Internal Probes.

Based on multiple sequence alignments, regions of the tuf gene unique tostaphylococci were identified. Staphylococcus-specific PCR primersTStaG422 (SEQ ID NO. 553) and TStaG765 (SEQ ID NO. 575) were derivedfrom these regions (Table 50). These PCR primers are displaced by twonucleotide positions compared to original Staphylococcus-specific PCRprimers described in our patent publication WO98/20157 (SEQ ID NOs. 17and 20 in the said patent publication). These modifications were done toensure specificity and ubiquity of the primer pair, in the light of newtuf sequence data revealed in the present patent application for severaladditional staphylococcal species and strains.

Similarly, sequence alignment analysis were performed to design genusand species-specific internal probes (see Tables 51 to 54). Two internalprobes specific for Staphylococcus (SEQ ID NOs. 605-606), five specificfor S. aureus (SEQ ID NOs. 584-588), five specific for S. epidermidis(SEQ ID NO. 589-593), two specific for S. haemolyticus (SEQ ID NOs.594-595), three specific for S. hominis (SEQ ID NOs. 596-598), fourspecific for S. saprophyticus (SEQ ID NOs. 599-601 and 695), and twospecific for coagulase-negative Staphylococcus species including S.epidermidis, S. hominis, S. saprophyticus, S. auricularis, S. capitis,S. haemolyticus, S. lugdunensis, S. simulans, S. cohnii and S. warneri(SEQ ID NOs. 1175-1176) were designed. The range of mismatches betweenthe Staphylococcus-specific 371-bp amplicon and each of the 20-merspecies-specific internal probes was from 1 to 5, in the middle of theprobe when possible. No mismatches were present in the twoStaphylococcus-specific probes for the 11 species analyzed: S. aureus,S. auricularis, S. capitis, S. cohnii, S. epidermidis, S. haemolyticus,S. hominis, S. lugdunensis, S. saprophyticus, S. simulans and S.warneri. In order to verify the intra-specific sequence conservation ofthe nucleotide sequence, sequences were obtained for the 371-bp ampliconfrom five unrelated ATCC and clinical strains for each of the species S.aureus, S. epidermidis, S. haemolyticus, S. hominis and S.saprophyticus. The Oligo™ (version 5.0) primer analysis software(National Biosciences, Plymouth, Minn.) was used to confirm the absenceof self-complementary regions within and between the primers or probes.When required, the primers contained inosines or degenerated nucleotidesat one or more variable positions. Oligonucleotide primers and probeswere synthesized on a model 394 DNA synthesizer (Applied Biosystems,Mississauga, Ontario, Canada). Detection of the hybridization wasperformed with the DIG-labeled dUTP incorporated during amplificationwith the Staphylococcus-specific PCR assay, and the hybridization signalwas detected with a luminometer (Dynex Technologies) as described abovein the section on luminescent detection of amplification products.Tables 51 to 54 illustrate the strategy for the selection of severalinternal probes.

PCR Amplification.

For all bacterial species, amplification was performed from purifiedgenomic DNA or from a bacterial suspension whose turbidity was adjustedto that of a 0.5 McFarland standard, which corresponds to approximately1.5×10⁸ bacteria per ml. One nanogram of genomic DNA or 1 □l of thestandardized bacterial suspension was transferred directly to a 19 □lPCR mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.2 □M (each) of the twoStaphylococcus genus-specific primers (SEQ ID NOs. 553 and 575), 200 □M(each) of the four deoxynucleoside triphosphates (Pharmacia Biotech),3.3 □g/□l bovine serum albumin (BSA) (Sigma-Aldrich Canada Ltd,Oakville, Ontario, Canada), and 0.5 U Taq polymerase (Promega) coupledwith TaqStart™ Antibody (Clontech). The PCR amplification was performedas follows: 3 min. at 94° C. for initial denaturation, then forty cyclesof two steps consisting of 1 second at 95° C. and 30 seconds at 55° C.,plus a terminal extension at 72° C. for 2 minutes. Detection of the PCRproducts was made by electrophoresis in agarose gels (2%) containing0.25 μg/ml of ethidium bromide. Visualization of the PCR products wasmade under UV at 254 nm.

For determination of the sensitivities of the PCR assays, two-folddilutions of purified genomic DNA were used to determine the minimalnumber of genome copies which can be detected.

Results

Amplifications with the Staphylococcus Genus-Specific PCR Assay.

The specificity of the assay was assessed by performing 30-cycle and40-cycle PCR amplifications with the panel of gram-positive (47 speciesfrom 8 genera) and gram-negative (33 species from 22 genera) bacterialspecies listed in Table 12. The PCR assay was able to detect efficiently27 of 27 staphylococcal species tested in both 30-cycle and 40-cycleregimens. For 30-cycle PCR, all bacterial species tested other thanstaphylococci were negative. For 40-cycle PCR, Enterococcus faecalis andMacrococcus caseolyticus were slightly positive for theStaphylococcus-specific PCR assay. The other species tested remainednegative. Ubiquity tests performed on a collection of 295 clinicalisolates provided by the microbiology laboratory of the CentreHospitalier Universitaire de Québec, Pavillon Centre Hospitalier del'Université Laval (CHUL), including Staphylococcus aureus (n=34), S.auricularis (n=2), S. capitis (n=19), S. cohnii (n=5), S. epidermidis(n=18), S. haemolyticus (n=21), S. hominis (n=73), S. lugdunensis(n=17), S. saprophyticus (n=6), S. simulans (n=3), S. warneri (n=32) andStaphylococcus sp. (n=65), showed a uniform amplification signal withthe 30-cycle PCR assays and a perfect relation between the genotype andclassical identification schemes.

The sensitivity of the Staphylococcus-specific assay with 30-cycle and40-cycle PCR protocols was determined by using purified genomic DNA fromthe 11 staphylococcal species previously mentioned. For PCR with 30cycles, a detection limit of 50 copies of genomic DNA was consistentlyobtained. In order to enhance the sensitivity of the assay, the numberof cycles was increased. For 40-cycle PCR assays, the detection limitwas lowered to a range of 5-10 genome copies, depending on thestaphylococcal species tested.

Hybridization Between the Staphylococcus-Specific 371-Bp Amplicon andSpecies-Specific or Genus-Specific Internal Probes.

Inter-species polymorphism was sufficient to generate species-specificinternal probes for each of the principal species involved in humandiseases (S. aureus, S. epidermidis, S. haemolyticus, S. hominis and S.saprophyticus). In order to verify the intra-species sequenceconservation of the nucleotide sequence, sequence comparisons wereperformed on the 371-bp amplicon from five unrelated ATCC and clinicalstrains for each of the 5 principal staphylococcal species: S. aureus,S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus.Results showed a high level of conservation of nucleotide sequencebetween different unrelated strains from the same species. This sequenceinformation allowed the development of staphylococcal speciesidentification assays using species-specific internal probes hybridizingto the 371-bp amplicon. These assays are specific and ubiquitous forthose five staphylococcal species. In addition to the species-specificinternal probes, the genus-specific internals probes were able torecognize all or most Staphylococcus species tested.

Example 8

Differentiating Between the Two Closely Related Yeast Species Candidaalbicans and Candida dubliniensis.

It is often useful for the clinician to be able to differentiate betweentwo very closely related species of microorganisms. Candida albicans isthe most important cause of invasive human mycose. In recent years, avery closely related species, Candida dubliniensis, was isolated inimmunosuppressed patients. These two species are difficult todistinguish by classic biochemical methods. This example demonstratesthe use of tuf sequences to differentiate Candida albicans and Candidadubliniensis. PCR primers SEQ ID NOs. 11-12, from previous patentpublication WO98/20157, were selected for their ability to specificallyamplify a tuf (elongation factor 1 alpha type) fragment from bothspecies (see Table 49 for primer positions). Within this tuf fragment, aregion differentiating C. albicans and C. dubliniensis by twonucleotides was selected and used to design two internal probes (seeTable 49 for probe design, SEQ ID NOs. 577 and 578) specific for eachspecies. Amplification of genomic DNA from C. albicans and C.dubliniensis was carried out using DIG-11-dUTP as described above in thesection on chemiluminescent detection of amplification products.Internal probes SEQ ID NOs. 577 and 578 were immobilized on the bottomof individual microtiter plates and hybridization was carried out asdescribed above in the above section on chemiluminescent detection ofamplification products. Luminometer data showed that the amplicon fromC. albicans hybridized only to probe SEQ ID NO. 577 while the ampliconfrom C. dubliniensis hybridized only to probe SEQ ID NO. 578, therebydemonstrating that each probe was species-specific.

Example 9

Specific Identification of Entamoeba histolytica.

Upon analysis of tuf (elongation factor 1 alpha) sequence data, it waspossible to find four regions where Entamoeba histolytica sequencesremained conserved while other parasitical and eukaryotic species havediverged. Primers TEntG38 (SEQ ID NO. 703), TEntG442 (SEQ ID NO. 704),TEntG534 (SEQ ID NO. 705), and TEntG768 (SEQ ID NO. 706) were designedso that SEQ ID NO. 703 could be paired with the three other primers. OnPTC-200 thermocyclers (MJ Research), the cycling conditions for initialsensitivity and specificity testing were 3 min. at 94° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 55° C., followed by terminal extension at 72°C. for 2 minutes. Detection of the PCR products was made byelectrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidiumbromide. The three primer pairs could detect the equivalent of less than200 E. histolytica genome copies. Specificity was tested using 0.5 ng ofpurified genomic DNA from a panel of microorganisms including Babesiabovis, Babesia microtti, Candida albicans, Crithidia fasciculata,Leishmania major, Leishmania hertigi and Neospora caninum. Only E.histolytica DNA could be amplified, thereby suggesting that the assaywas species-specific.

Example 10

Sensitive Identification of Chlamydia trachomatis.

Upon analysis of tuf sequence data, it was possible to find two regionswhere Chlamydia trachomatis sequences remained conserved while otherspecies have diverged. Primers Ctr82 (SEQ ID NO. 554) and Ctr249 (SEQ IDNO. 555) were designed. With the PTC-200 thermocyclers (MJ Research),the optimal cycling conditions for maximum sensitivity and specificitywere determined to be 3 min. at 94° C. for initial denaturation, thenforty cycles of two steps consisting of 1 second at 95° C. and 30seconds at 60° C., followed by terminal extension at 72° C. for 2minutes. Detection of the PCR products was made by electrophoresis inagarose gels (2%) containing 0.25 μg/ml of ethidium bromide. The assaycould detect the equivalent of 8 C. trachomatis genome copies.Specificity was tested with 0.1 ng of purified genomic DNA from a panelof microorganisms including 22 species commonly encountered in thevaginal flora (Bacillus subtilis, Bacteroides fragilis, Candidaalbicans, Clostridium difficile, Corynebacterium cervicis,Corynebacterium urealyticum, Enterococcus faecalis, Enterococcusfaecium, Escherichia coli, Fusobacterium nucleatum, Gardnerellavaginalis, Haemophilus influenzae, Klebsiella oxytoca, Lactobacillusacidophilus, Peptococcus niger, Peptostreptococcus prevotii,Porphyromonas asaccharolytica, Prevotella melaninogenica,Propionibacterium acnes, Staphylococcus aureus, Streptococcusacidominimus, and Streptococcus agalactiae). Only C. trachomatis DNAcould be amplified, thereby suggesting that the assay wasspecies-specific.

Example 11

Genus-Specific Detection and Identification of Enterococci.

Upon analysis of tuf sequence data and comparison with the repertory oftuf sequences, it was possible to find two regions where Enterococcussequences remained conserved while other genera have diverged (Table55). Primer pair Encg313dF and Encg599c (SEQ ID NOs. 1137 and 1136) wastested for its specificity by using purified genomic DNA from a panel ofbacteria listed in Table 10. Using the PTC-200 thermocycler (MJResearch), the optimal cycling conditions for maximum sensitivity andspecificity were determined to be 3 min. at 94° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 55° C., followed by terminal extension at 72°C. for 2 minutes. Detection of the PCR products was made byelectrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidiumbromide. Visualization of the PCR products was made under UV at 254 nm.The 18 enterococcal species listed in Table 10 were all amplifiedefficiently. The only other species amplified were Abiotrophia adiacens,Gemella haemolysans and Gemella morbillorum, three gram-positivespecies. Sensitivity tested with several strains of E. casseliflavus, E.faecium, E. faecalis, E. flavescens and E. gallinarum and with onestrain of each other Enterococcus species listed in Table 10 ranged from1 to 10 copies of genomic DNA. The sequence variation within the 308-bpamplicon was sufficient so that internal probes could be used tospeciate the amplicon and differenciate enterococci from Abiotrophiaadiacens, Gemella haemolysans and Gemella morbillorum, thereby allowingto achieve excellent specificity. Species-specific internal probes weregenerated for each of the clinically important species, E. faecalis (SEQID NO. 1174), E. faecium (SEQ ID NO. 602), and the group including E.casseliflavus, E. flavescens and E. gallinarum (SEQ ID NO. 1122) (Table56). The species-specific internal probes were able to differentiatetheir respective Enterococcus species from all other Enterococcusspecies. These assays are sensitive, specific and ubiquitous for thosefive Enterococcus species.

Example 12

Identification of the Major Bacterial Platelets Contaminants Using tufSequences with a Multiplex PCR Test.

Blood platelets preparations need to be monitored for bacterialcontaminations. The tuf sequences of 17 important bacterial contaminantsof platelets were aligned. As shown in Table 57, analysis of thesesequences allowed the design of PCR primers. Since in the case ofcontamination of platelet concentrates, detecting all species (not justthe more frequently encountered ones) is desirable, perfect specificityof primers was not an issue in the design. However, sensitivity isimportant. That is why, to avoid having to put too much degeneracy, onlythe most frequent contaminants were included in primer design, knowingthat the selected primers would anyway be able to amplify more speciesthan the 17 used in the design because they target highly conservedregions of tuf sequences. Oligonucleotide sequences which are conservedin these 17 major bacterial contaminants of platelet concentrates werechosen (oligos Tplaq 769 and Tplaq 991, respectively SEQ ID NOs. 636 and637) thereby permitting the detection of these bacterial species.However, sensitivity was slightly deficient with staphylococci. Toensure maximal sensitivity in the detection of all the more frequentbacterial contaminants, a multiplex assay also including oligonucleotideprimers targeting the Staphylococcus genera (oligos Stag 422, SEQ ID NO.553; and Stag 765, SEQ ID NO. 575) was developed. The bacterial speciesdetected with the assay are listed in Table 14.

The primer pairs, oligos SEQ ID NO. 636 and SEQ ID NO. 637 that give anamplification product of 245 pb, and oligos SEQ ID NO. 553 and SEQ IDNO. 575 that give an amplification product of 368 pb, were usedsimultaneously in the multiplex PCR assay. Detection of these PCRproducts was made on the LightCycler thermocycler (Idaho Technology)using SYBR® Green I (Molecular Probe Inc.). SYBR® Green I is afluorescent dye that binds specifically to double-stranded DNA.

Fluorogenic detection of PCR products with the LightCycler was carriedout using 1.0 μM of both Tplaq primers (SEQ ID NOs. 636-637) and 0.4 μMof both TStaG primers (SEQ ID NOs. 553 and 575), 2.5 mM MgCl₂, BSA 7.5μM, dNTP 0.2 mM (Pharmacia), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.5 UTaq DNA polymerase (Boerhinger Mannheim) coupled with TaqStart™ antibody(Clontech), and 0.07 ng of genomic DNA sample in a final volume of 7 μl.The optimal cycling conditions for maximum sensitivity and specificitywere 1 minute at 94° C. for initial denaturation, then forty-five cyclesof three steps consisting of 0 second at 95° C., 5 seconds at 60° C. and9 seconds at 72° C. Amplification was monitored during each elongationcycle by measuring the level of SYBR® Green I. However, real analysistakes place after PCR. Melting curves are done for each sample andtransformation of the melting peak allows determination of Tm. Thusprimer-dimer and specific PCR product are discriminated. With thisassay, all prominent bacterial contaminants of platelet concentrateslisted in Table 57 and Table 14 were detected. Sensitivity tests wereperformed on the 9 most frequent bacterial contaminants of platelets.The detection limit was less than 20 genome copies for E. cloacae, B.cereus, S. choleraesuis and S. marcescens; less than 15 genome copiesfor P. aeruginosa; and 2 to 3 copies were detected for S. aureus, S.epidermidis, E. coli and K. pneumoniae. Further refinements of assayconditions should increase sensitivity levels.

Example 13

The Resolving Power of the tuf and atpD Sequences Databases isComparable to the Biochemical Methods for Bacterial Identification.

The present gold standard for bacterial identification is mainly basedon key morphological traits and batteries of biochemical tests. Here wedemonstrate that the use of tuf and atpD sequences combined with simplephylogenetic analysis of databases formed by these sequences iscomparable to the gold standard. In the process of acquiring data forthe tuf sequences, we sequenced the tuf gene of a strain that was givento us labelled as Staphylococcus hominis ATCC 35982. That tuf sequence(SEQ ID NO. 192) was incorporated into the tuf sequences database andsubjected to a basic phylogenic analysis using the Pileup command fromversion 10 of the GCG package (Genetics Computer Group). This analysisindicated that SEQ ID NO. 192 is not associated with other S. hominisstrains but rather with the S. warneri strains. The ATCC 35982 strainwas sent to the reference laboratory of the Laboratoire de santépublique du Québec (LSPQ). They used the classic identification schemefor staphylococci (Kloos and Schleifer, 1975., J. Clin. Microbiol.1:82-88). Their results shown that although the colonial morphologycould correspond to S. hominis, the more precise biochemical assays didnot. These assays included discriminant mannitol, mannose and riboseacidification tests as well as rapid and dense growth in deepthioglycolate agar. The LSPQ report identified strain ATCC 35982 as S.warneri which confirms our database analysis. The same thing happenedfor S. warneri (SEQ ID NO. 187) which had initially been identified asS. haemolyticus by a routine clinical laboratory using a low resolvingpower automated system (MicroScan, AutoScan-4™). Again, the tuf and LSPQanalysis agreed on its identification as S. warneri. In numerous otherinstances, in the course of acquiring tuf and atpD sequence data fromvarious species and genera, analysis of our tuf and/or atpD sequencedatabases permitted the exact identification of mislabelled orerroneously identified strains. These results clearly demonstrate theusefulness and the high resolving power of our sequence-basedidentification assays using the tuf and atpD sequences databases.

Example 14

Detection of Group B Streptococci from Clinical Specimens.*

Introduction

Streptococcus agalactiae, the group B streptococcus (GB S), isresponsible for a severe illness affecting neonate infants. Thebacterium is passed from the healthy carrier mother to the baby duringdelivery. To prevent this infection, it is recommended to treatexpectant mothers susceptible of carrying GBS in their vaginal/analflora. Carrier status is often a transient condition and rigorousmonitoring requires cultures and classic bacterial identification weeksbefore delivery. To improve the detection and identification of GBS wedeveloped a rapid, specific and sensitive PCR test fast enough to beperformed right at delivery.

Materials and Methods

GBS Clinical Specimens.

A total of 66 duplicate vaginal/anal swabs were collected from 41consenting pregnant women admitted for delivery at the CentreHospitalier Universitaire de Québec, Pavillon Saint-Francois d'Assisefollowing the CDC recommendations. The samples were obtained eitherbefore or after rupture of membranes. The swab samples were tested atthe Centre de Recherche en Infectiologie de l'Université Laval within 24hours of collection. Upon receipt, one swab was cut and then the tip ofthe swab was added to GNS selective broth for identification of group Bstreptococci (GBS) by the standard culture methods recommended by theCDC. The other swab was processed following the instruction of the IDIDNA extraction kit (Infectio Diagnotics (IDI) Inc.) prior to PCRamplification.

Oligonucleotides.

PCR primers, Tsag340 (SEQ ID NO. 549) and Tsag552 (SEQ ID NO. 550)complementary to the regions of the tuf gene unique for GBS weredesigned based upon a multiple sequence alignment using our repertory oftuf sequences. Oligo primer analysis software (version 5.0) (NationalBiosciences) was used to analyse primers annealing temperature,secondary structure potential as well as mispriming and dimerizationpotential. The primers were synthesized using a model 391 DNAsynthesizer (Applied Biosystems).

A pair of fluorescently labeled adjacent hybridization probes Sag465-F(SEQ ID NO. 583) and Sag436-C(SEQ ID NO. 582) were synthesized andpurified by Operon Technologies. They were designed to meet therecommendations of the manufacturer (Idaho Technology) and based uponmultiple sequence alignment analysis using our repertory of tufsequences to be specific and ubiquitous for GBS. These adjacent probes,which are separated by one nucleotide, allow fluorescence resonanceenergy transfer (FRET), generating an increased fluorescence signal whenboth hybridized simultaneously to their target sequences. The probe SEQID NO. 583 was labeled with FITC in 3 prime while SEQ ID NO. 582 waslabeled with Cy5 in 5 prime. The Cy5-labeled probes contained a3′-blocking phosphate group to prevent extension of the probes duringthe PCR reactions.

PCR Amplification.

Conventional amplifications were performed either from 2 μl of apurified genomic DNA preparation or cell lysates of vaginal/analspecimens. The 20 μl PCR mixture contained 0.4 μM of each GB S-specificprimer (SEQ ID NOs. 549-550), 200 μM of each deoxyribonucleotide(Pharmacia Biotech), 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% TritonX-100, 2.5 mM MgCl₂, 3.3 mg/ml bovine serum albumin (BSA) (Sigma), and0.5 U of Taq polymerase (Promega) combined with the TaqStart™ antibody(Clontech). The TaqStart™ antibody, which is a neutralizing monoclonalantibody of Taq DNA polymerase, was added to all PCR reactions toenhance the efficiency of the amplification. The PCR mixtures weresubjected to thermal cycling (3 min at 95° C. and then 40 cycles of 1 sat 95° C., and 30 s at 62° C. with a 2-min final extension at 72° C.)with a PTC-200 DNA Engine thermocycler (MJ research). The PCR-amplifiedreaction mixture was resolved by agarose gel electrophoresis.

The LightCycler™ PCR amplifications were performed with 1 μl of apurified genomic DNA preparation or cell lysates of vaginal/analspecimens. The 100 amplification mixture consisted of 0.4 μM eachGBS-specific primer (SEQ ID NOs. 549-550), 200 μM each dNTP, 0.2 μM eachfluorescently labeled probe (SEQ ID NOs. 582-583), 300 μg/ml BSA(Sigma), and 1 μl of 10×PC2 buffer (containing 50 mM Tris-HCl (pH 9.1),16 mM ammonium sulfate, 3.5 mM Mg²⁺, and 150 μg/ml BSA) and 0.5 UKlenTaq1™ (AB Peptides) coupled with TaqStart™ antibody (Clontech).KlenTaq1™ is a highly active and more heat-stable DNA polymerase without5′-exonuclease activity. This prevents hydrolysis of hybridized probesby the 5′ to 3′ exonuclease activity. A volume of 7 μl of the PCRmixture was transferred into a composite capillary tube (IdahoTechnology). The tubes were then centrifuged to move the reactionmixture to the tips of the capillaries and then cleaned withoptical-grade methanol. Subsequently the capillaries were loaded intothe carousel of a LC32 LightCycler™ (Idaho Technology), an instrumentthat combines rapid-cycle PCR with fluorescence analysis for continuousmonitoring during amplification. The PCR reaction mixtures weresubjected to a denaturation step at 94° C. for 3 min followed by 45cycles of 0 s at 94° C., 20 s at 64° C. and 10 s at 72° C. with atemperature transition rate of 20° C./s. Fluorescence signals wereobtained at each cycle by sequentially positioning each capillary on thecarousel at the focus of optical elements affiliated to the built-influorimeter for 100 milliseconds. Complete amplification and analysisrequired about 35 min.

Specificity and Sensitivity Tests.

The specificity of the conventional and LightCycler™ PCR assays wasverified by using purified genomic DNA (0.1 ng/reaction) from a batteryof ATCC reference strains representing 35 clinically relevantgram-positive species (Abiotrophia defectiva ATCC 49176, Bifidobacteriumbreve ATCC 15700, Clostridium difficile ATCC 9689, Corynebacteriumurealyticum ATCC 43042, Enterococcus casseliflavus ATCC 25788,Enterococcus durans ATCC 19432, Enterococcus faecalis ATCC 29212,Enterococcus faecium ATCC 19434, Enterococcus gallinarum ATCC 49573,Enterococcus raffinosus ATCC 49427, Lactobacillus reuteri ATCC 23273,Lactococcus lactis ATCC 19435, Listeria monocytogenes ATCC 15313,Peptococcus niger ATCC 27731, Peptostreptococcus anaerobius ATCC 27337,Peptostreptococcus prevotii ATCC 9321, Staphylococcus aureus ATCC 25923,Staphylococcus epidermidis ATCC 14990, Staphylococcus haemolyticus ATCC29970, Staphylococcus saprophyticus ATCC 15305, Streptococcus agalactiaeATCC 27591, Streptococcus anginosus ATCC 33397, Streptococcus bovis ATCC33317, Streptococcus constellatus ATCC 27823, Streptococcus dysgalactiaeATCC 43078, Streptococcus gordonii ATCC 10558, Streptococcus mitis ATCC33399, Streptococcus mutans ATCC 25175, Streptococcus oralis ATCC 35037,Streptococcus parauberis ATCC 6631, Streptococcus pneumoniae ATCC 6303,Streptococcus pyogenes ATCC 19615, Streptococcus salivarius ATCC 7073,Streptococcus sanguinis ATCC 10556, Streptococcus uberis ATCC 19436).These microbial species included 15 species of streptococci and manymembers of the normal vaginal and anal floras. In addition, 40 GBSisolates of human origin, whose identification was confirmed by a latexagglutination test (Streptex, Murex), were also used to evaluate theubiquity of the assay.

For determination of the sensitivities (i.e., the minimal number ofgenome copies that could be detected) for conventional and LightCycler™PCR assays, serial 10-fold or 2-fold dilutions of purified genomic DNAfrom 5 GBS ATCC strains were used.

Results

Evaluation of the GBS-Specific Conventional and LightCycler™ PCR Assays.

The specificity of the two assays demonstrated that only DNAs from GBSstrains could be amplified. Both PCR assays did not amplify DNAs fromany other bacterial species tested including 14 streptococcal speciesother than GBS as well as phylogenetically related species belonging tothe genera Enterococcus, Peptostreptococcus and Lactococcus. Importantmembers of the vaginal or anal flora, including coagulase-negativestaphylococci, Lactobacillus sp., and Bacteroides sp. were also negativewith the GB S-specific PCR assay. The LightCycler™ PCR assays detectedonly GBS DNA by producing an increased fluorescence signal which wasinterpreted as a positive PCR result. Both PCR methods were able toamplify all of 40 GBS clinical isolates, showing a perfect correlationwith the phenotypic identification methods.

The sensitivity of the assay was determined by using purified genomicDNA from the 5 ATCC strains of GBS. The detection limit for all of these5 strains was one genome copy of GB S. The detection limit of the assaywith the LightCycler™ was 3.5 fg of genomic DNA (corresponding to 1-2genome copies of GBS). These results confirmed the high sensitivity ofour GBS-specific PCR assay.

Direct Detection of GBS from Vaginal/Anal Specimens.

Among 66 vaginal/anal specimens tested, 11 were positive for GBS by bothculture and PCR. There was one sample positive by culture only. Thesensitivity of both PCR methods with vaginal/anal specimens foridentifying colonization status in pregnant women at delivery was 91.7%when compared to culture results. The specificity and positivepredictive values were both 100% and the negative predictive value was97.8%. The time for obtaining results was approximately 45 min forLightCycler™ PCR, approximately 100 min for conventional PCR and 48hours for culture.

Conclusion

We have developed two PCR assays (conventional and LightCycler™) for thedetection of GBS, which are specific (i.e., no amplification of DNA froma variety of bacterial species other than GBS) and sensitive (i.e., ableto detect around 1 genome copy for several reference ATCC strains ofGBS). Both PCR assays are able to detect GBS directly from vaginal/analspecimens in a very short turnaround time. Using the real-time PCR assayon LightCycler™, we can detect GBS carriage in pregnant women atdelivery within 45 minutes.

Example 15

Simultaneous Detection and Identification of Streptococcus pyogenes andits Pyrogenic Exotoxin A.

The rapid detection of Streptococcus pyogenes and of its pyrogenicexotoxin A is of clinical importance. We developed a multiplex assaywhich permits the detection of strains of S. pyogenes carrying thepyrogenic toxin A gene, which is associated with scarlet fever and otherpathologies. In order to specifically detect S. pyogenes, nucleotidesequences of the pyrrolidone carboxylyl peptidase (pcp) gene werealigned to design PCR primers Spy291 (SEQ ID NO. 1211) and Spy473 (SEQID NO. 1210). Next, we designed primers for the specific detection ofthe pyrogenic exotoxin A. Nucleotide sequences of the speA gene, carriedon the bacteriophage T12, were aligned as shown in Table 61 to designPCR primers Spytx814 (SEQ ID NO. 994) and Spytx 927 (SEQ ID NO. 995).

The primer pairs: oligos SEQ ID NOs. 1210-1211, yielding anamplification product of 207 bp, and oligos SEQ ID NOs. 994-995,yielding an amplification product of 135 bp, were used in a multiplexPCR assay.

PCR amplification was carried out using 0.4 μM of both pairs of primers,2.5 mM MgCl₂, BSA 0.05 μM, dNTP 0.2 μM (Pharmacia), 10 mM Tris-HCl (pH9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.5 U Taq DNA polymerase(Promega) coupled with TaqStart™ antibody (Clontech Laboratories Inc.),and 1 μl of genomic DNA sample in a final volume of 20 μl. PCRamplification was performed using a PTC-200 thermal cycler (MJResearch). The optimal cycling conditions for maximum specificity andsensitivity were 3 minutes at 94° C. for initial denaturation, thenforty cycles of two steps consisting of 1 second at 95° C. and 30seconds at 63° C., followed by a final step of 2 minutes at 72° C.Detection of the PCR products was made by electrophoresis in agarosegels (2%) containing 0.25 μg/ml of ethidium bromide. Visualization ofthe PCR products was made under UV at 254 nm.

The detection limit was less than 5 genome copies for both S. pyogenesand its pyrogenic exotoxin A. The assay was specific for pyrogenicexotoxin A-producing S. pyogenes: strains of the 27 other species ofStreptococcus tested, as well as 20 strains of various gram-positive andgram-negative bacterial species were all negative.

A similar approach was used to design an alternative set ofspeA-specific primers (SEQ ID NOs. 996 to 998, see Table 62). Inaddition, another set of primers based on the tuf gene (SEQ ID NOs. 999to 1001, see Table 63) could be used to specifically detectStreptococcus pyogenes.

Example 16

Real-Time Detection and Identification of Shiga Toxin-ProducingBacteria.

Shiga toxin-producing Escherichia coli and Shigella dysenteriae causebloody diarrhea. Currently, identification relies mainly on thephenotypic identification of S. dysenteriae and E. coli serotypeO157:H7. However, other serotypes of E. coli are increasingly found tobe producers of type 1 and/or type 2 Shiga toxins. Two pairs of PCRprimers targeting highly conserved regions present in each of the Shigatoxin genes stx₁ and stx₂ were designed to amplify all variants of thosegenes (see Tables 64 and XXVII). The first primer pair, oligonucleotides1SLT224 (SEQ ID NO. 1081) and 1SLT385 (SEQ ID NO. 1080), yields anamplification product of 186 bp from the stx₁ gene. For this amplicon,the 1SLTB1-Fam (SEQ ID NO. 1084) molecular beacon was designed for thespecific detection of stx₁ using the fluorescent label6-carboxy-fluorescein. The 1SltS1-FAM (SEQ ID NO. 2012) molecularscorpion was also designed as an alternate way for the specificdetection of stx₁. A second pair of PCR primers, oligonucleotides2SLT537 (SEQ ID NO. 1078) and 2SLT678b (SEQ ID NO. 1079), yields anamplification product of 160 bp from the sbc₂ gene. Molecular beacon2SLTB1-Tet (SEQ ID NO. 1085) was designed for the specific detection ofstx₂ using the fluorescent label 5-tetrachloro-fluorescein. Both primerpairs were combined in a multiplex PCR assay.

PCR amplification was carried out using 0.8 μM of primer pair SEQ IDNOs. 1080-1081, 0.5 μM of primer pair SEQ ID NOs. 1078-1079, 0.3 μM ofeach molecular beacon, 8 mM MgCl₂, 490 μg/mL BSA, 0.2 mM dNTPs(Pharmacia), 50 mM Tris-HCl, 16 mM NH₄SO₄, 1×TaqMaster (Eppendorf), 2.5U KlenTaq1 DNA polymerase (AB Peptides) coupled with TaqStart™ antibody(Clontech Laboratories Inc.), and 1 μl of genomic DNA sample in a finalvolume of 25 PCR amplification was performed using a SmartCycler thermalcycler (Cepheid). The optimal cycling conditions for maximum sensitivityand specificity were 60 seconds at 95° C. for initial denaturation, then45 cycles of three steps consisting of 10 seconds at 95° C., 15 secondsat 56° C. and 5 seconds at 72° C. Detection of the PCR products was madein real-time by measuring the fluorescent signal emitted by themolecular beacon when it hybridizes to its target at the end of theannealing step at 56° C.

The detection limit was the equivalent of less than 5 genome copies. Theassay was specific for the detection of both toxins, as demonstrated bythe perfect correlation between PCR results and the phenotypiccharacterization performed using antibodies specific for each Shigatoxin type. The assay was successfully performed on several Shigatoxin-producing strains isolated from various geographic areas of theworld, including 10 O157:H7 E. coli, 5 non-O157:H7 E. coli and 4 S.dysenteriae.

Example 17

Development of a PCR Assay for the Detection and Identification ofStaphylococci at Genus and Species Levels and its Associated mecA Gene.

The Staphylococcus-specific PCR primers described in Example 7 (SEQ IDNOs. 553 and 575) were used in multiplex with the mecA-specific PCRprimers and the S. aureus-specific primers described in our assignedU.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and 262 for mecA and SEQ IDNOs. 152 and 153 for S. aureus in the said patent). Sequence alignmentanalysis of 10 publicly available mecA gene sequences allowed to designan internal probe specific to mecA (SEQ ID NO. 1177). An internal probewas also designed for the S. aureus-specific amplicon (SEQ ID NO 1234).PCR amplification and agarose gel electrophoresis of the amplifiedproducts were performed as described in Example 7, with the exceptionthat 0.4 μM (each) of the two Staphylococcus-specific primers (SEQ IDNOs. 553 and 575) and 0.4 μM (each) of the mecA-specific primers and 0.4μM (each) of the S. aureus-specific primers were used in the PCRmixture. The specificity of the multiplex assay with 40-cycle PCRprotocols was verified by using purified genomic DNA from fivemethicillin-resistant and fifteen methicillin-sensitive staphylococcalstrains. The sensitivity of the multiplex assay with 40-cycle PCRprotocols was determined by using purified genomic DNA from twenty-threemethicillin-resistant and twenty-eight methicillin-sensitivestaphylococcal strains. The detection limit was 2 to 10 genome copies ofgenomic DNA, depending on the staphylococcal species tested.Furthermore, the mecA-specific internal probe, the S. aureus-specificinternal probe and the coagulase-negative staphylococci-specificinternal probe (described in Example 7) were able to recognizetwenty-three methicillin-resistant staphylococcal strains andtwenty-eight methicillin-sensitive staphylococcal strains with highsensitivity and specificity.

The format of the assay is not limited to the one described above. Aperson skilled in the art could adapt the assay for different formatssuch as PCR with real-time detection using molecular beacon probes.Molecular beacon probes designed to be used in this assay include, butare not limited to, SEQ ID NO. 1232 for detection of the S.aureus-specific amplicon, SEQ ID NO. 1233 for detection ofcoagulase-negative staphylococci and SEQ ID NO. 1231 for detection ofmecA.

Alternatively, a multiplex PCR assay containing theStaphylococcus-specific PCR primers described in Example 7 (SEQ ID NOs.553 and 575) and the mecA-specific PCR primers described in our assignedU.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and 262 in the said patent)were developed. PCR amplification and agarose gel electrophoresis of theamplified products were performed as described in Example 7, with theexception that 0.4 μM (each) of the Staphylococcus-specific primers (SEQID NOs. 553 and 575) and 0.4 μM (each) of the mecA-specific primersdescribed in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and262 in the said patent) were used in the PCR mixture. The sensitivity ofthe multiplex assay with 40-cycle PCR protocols was determined by usingpurified genomic DNA from two methicillin-resistant and fivemethicillin-sensitive staphylococcal strains. The detection limit was 2to 5 copies of genomic DNA, depending on the staphylococcal speciestested. The specificity of the multiplex PCR assay coupled withcapture-probe hybridization was tested with two strains ofmethicillin-resistant S. aureus, two strains of methicillin-sensitive S.aureus and seven strains of methicillin-sensitive coagulase-negativestaphylococci. The mecA-specific internal probe (SEQ ID NO. 1177) andthe S. aureus-specific internal probe (SEQ ID NO. 587) described inExample 7 were able to recognize all the strains with high specificityshowing a perfect correlation with susceptibility to methicillin. Thesensitivity of the PCR assay coupled with capture-probe hybridizationwas tested with one strain of methicillin-resistant S. aureus. Thedetection limit was around 10 copies of genomic DNA.

Example 18

Sequencing of pbp1a, pbp2b and pbp2x Genes of Streptoccoccus pneumoniae.

Penicillin resistance in Streptococcus pneumoniae involves thesequential alteration of up to five penicillin-binding proteins (PBPs)1A, 1B, 2A, 2X and 2B in such a way that their affinity is greatlyreduce toward the antibiotic molecule. The altered PBP genes have arisenas the result of interspecies recombination events from relatedstreptococcal species. Among the PBPs usually found in S. pneumoniae,PBPs 1A, 2B, and 2X play the most important role in the development ofpenicillin resistance. Alterations in PBP 2B and 2X mediate low-levelresistance to penicillin while additional alterations in PBP 1A plays asignificant role in full penicillin resistance.

In order to generate a database for pbp sequences that can be used fordesign of primers and/or probes for the specific and ubiquitousdetection of β-lactam resistance in S. pneumoniae, pbp1a, pbp2b andpbp2×DNA fragments sequenced by us or selected from public databases(GenBank and EMBL) from a variety of S. pneumoniae strains were used todesign oligonucleotide primers. This database is essential for thedesign of specific and ubiquitous primers and/or probes for detection ofβ-lactam resistance in S. pneumoniae since the altered PBP 1A, PBP 2Band PBP 2X of β-lactam resistant S. pneumoniae are encoded by mosaicgenes with numerous sequence variations among resistant isolates. ThePCR primers were located in conserved regions of pbp genes and were ableto amplify pbp1a, pbp2b, and pbp2x sequences of several strains of S.pneumoniae having various levels of resistance to penicillin andthird-generation cephalosporins. Using primer pairs SEQ ID NOs. 1125 and1126, SEQ ID NOs. 1142 and 1143, SEQ ID NOs. 1146 and 1147, it waspossible to amplify and determine pbp1a sequences SEQ ID NOs. 1004-1018,1648, 2056-2060 and 2062-2064, pbp2b sequences SEQ ID NOs. 1019-1033,and pbp2x sequences SEQ ID NOs. 1034-1048. Six other PCR primers (SEQ IDNOs. 1127-1128, 1144-1145, 1148-1149) were also designed and used tocomplete the sequencing of pbp1a, pbp2b and pbp2x amplificationproducts. The described primers (SEQ ID NOs. 1125 and 1126, SEQ ID NOs.1142 and 1143, SEQ ID NOs. 1146 and 1147, SEQ ID NOs. 1127-1128,1144-1145, 1148-1149) represent a powerful tool for generating new pbpsequences for design of primers and/or probes for detection of β-lactamresistance in S. pneumoniae.

Example 19

Sequencing of hexA Genes of Streptococcus Species.

The hexA sequence of S. pneumoniae described in our assigned U.S. Pat.No. 5,994,066 (SEQ ID NO. 31 in the said patent, SEQ ID NO. 1183 in thepresent application) allowed the design of a PCR primer (SEQ ID NO.1182) which was used with primer Spn1401 described in our assigned U.S.Pat. No. 5,994,066 (SEQ ID NO. 156 in the said patent, SEQ ID NO. 1179in the present application) to generate a database for hexA sequencesthat can be used to design primers and/or probes for the specificidentification and detection of S. pneumoniae (Table 80). Using primersSEQ ID NO. 1179 and SEQ ID NO. 1182 (Table 80), it was possible toamplify and determine the hexA sequence from S. pneumoniae (4 strains)(SEQ ID NOs. 1184-1187), S. mitis (three strains) (SEQ ID NOs.1189-1191) and S. oralis (SEQ ID NO. 1188).

Example 20

Development of Multiplex PCR Assays Coupled with Capture ProbeHybridization for the Detection and Identification of Streptococcuspneumoniae and its Penicillin Resistance Genes.

Two different assays were developed to identify S. pneumoniae and itssusceptibility to penicillin.

Assay I:

Bacterial Strains.

The specificity of the multiplex PCR assay was verified by using a panelof ATCC (American Type Culture Collection) reference strains consistingof 33 gram-negative and 67 gram-positive bacterial species (Table 13).In addition, a total of 98 strains of S. pneumoniae, 16 strains of S.mitis and 3 strains of S. oralis from the American Type CultureCollection, the microbiology laboratory of the Centre HospitalierUniversitaire de Québec, Pavillon Centre Hospitalier de l'UniversitéLaval (CHUL), (Ste-Foy, Québec, Canada), the Laboratoire de santépublique du Québec, (Sainte-Anne-de-Bellevue, Québec, Canada), theSunnybrook and Women's College Health Sciences Centre (Toronto, Canada),the Infectious Diseases Section, Department of Veterans Affairs MedicalCenter, (Houston, USA) were also tested to further validate theStreptococcus pneumoniae-specific PCR assay. The penicillin MICs(minimal inhibitory concentrations) were measured by the broth dilutionmethod according to the recommended protocol of NCCLS.

PCR Primers and Internal Probes.

The analysis of hexA sequences from a variety of streptococcal speciesfrom the publicly available hexA sequence and from the databasedescribed in Example 19 (SEQ ID NOs. 1184-1191) allowed the selection ofa PCR primer specific to S. pneumoniae, SEQ ID NO. 1181. This primer wasused with the S. pneumoniae-specific primer SEQ ID NO. 1179 to generatean amplification product of 241 bp (Table 80). The PCR primer SEQ ID NO.1181 is located 127 nucleotides downstream on the hexA sequence comparedto the original S. pneumoniae-specific PCR primer Spn1515 described inour assigned U.S. Pat. No. 5,994,066 (SEQ ID NO. 157 in the saidpatent). These modifications were done to ensure the design of the S.pneumoniae-specific internal probe according to the new hexA sequencesof several streptococcal species from the database described in Example19 (SEQ ID NOs. 1184-1191).

The analysis of pbp1a sequences from S. pneumoniae strains with variouslevels of penicillin resistance from public databases and from thedatabase described in Example 18 allowed the identification of aminoacid substitutions Ile-459 to Met and Ser-462 to Ala that occur inisolates with high-level penicillin resistance (MICs≥1 μg/ml), and aminoacid substitutions Ser-575 to Thr, Gln-576 to Gly and Phe-577 to Tyrthat are common to all penicillin-resistant isolates with MICs≥0.25μg/ml. As shown in Table 69, PCR primer pair SEQ ID NOs. 1130 and 1131were designed to detect high-level penicillin resistance (MICs≥1 μg/ml),whereas PCR primer pair SEQ ID NOs. 1129 and 1131 were designed todetect intermediate- and high-level penicillin resistance (MICs≥0.25μg/ml).

The analysis of hexA sequences from the publicly available hexA sequenceand from the database described in Example 19 allowed the design of aninternal probe specific to S. pneumoniae (SEQ ID NO. 1180) (Table 80).The range of mismatches between the S. pneumoniae-specific 241-bpamplicon was from 2 to 5, in the middle of the 19-bp probe. The analysisof pbp1a sequences from public databases and from the database describedin Example 18 allowed the design of five internal probes containing allpossible mutations to detect the high-level penicillin resistance 383-bpamplicon (SEQ ID NOs. 1197, 1217-1220). Alternatively, two otherinternal probes (SEQ ID NOs. 2024-2025) can also be used to detect thehigh-level penicillin resistance 383-bp amplicon. Five internal probescontaining all possible mutations to detect the 157-bp amplicon whichincludes intermediate- and high-level penicillin resistance were alsodesigned (SEQ ID NOs. 1094, 1192-1193, 1214 and 1216). Design andsynthesis of primers and probes, and detection of the probehybridization were performed as described in Example 7. Table 69illustrates one of the internal probe for detection of the high-levelpenicillin resistance 383-bp amplicon (SEQ ID NO. 1197) and one of theinternal probe for detection of the intermediate- and high-levelpenicillin resistance 157-bp amplicon (SEQ ID NO. 1193).

PCR Amplification.

For all bacterial species, amplification was performed from purifiedgenomic DNA using a PTC-200 thermocycler (MJ Research). 1 μl of genomicDNA at 0.1 ng/μ1, or 1 μl of a bacterial lysate, was transferred to a 19μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (H9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.1 μM (each) of the S.pneumoniae-specific primers SEQ ID NO. 1179 and SEQ ID NO. 1181, 0.2 μMof primer SEQ ID NO. 1129, 0.7 μM of primer SEQ ID NO. 1131, and 0.6 μMof primer SEQ ID NO. 1130, 0.05 mM bovine serum albumin (BSA), and 0.5 UTaq polymerase (Promega) coupled with TaqStart™ antibody. In order togenerate Digoxigenin (DIG)-labeled amplicons for capture probehybridization, 0.1×PCR DIG labeling four deoxynucleoside triphosphatesmix (Boehringer Mannheim GmbH) was used for amplification.

For determination of the sensitivity of the PCR assays, 10-folddilutions of purified genomic DNA were used to determine the minimalnumber of genome copies which can be detected.

Capture Probe Hybridization.

The DIG-labeled amplicons were hybridized to the capture probes bound to96-well plates. The plates were incubated with anti-DIG-alkalinephosphatase and the chemiluminescence was measured by using aluminometer (MLX, Dynex Technologies Inc.) after incubation with CSPDand recorded as Relative Light Unit (RLU). The RLU ratio of testedsample with and without captures probes was then calculated. A ratio≥2.0 was defined as a positive hybridization signal. All reactions wereperformed in duplicate.

Results

Amplifications with the Multiplex PCR Assay.

The specificity of the assay was assessed by performing 40-cycle PCRamplifications with the panel of gram-positive (67 species from 12genera) and gram-negative (33 species from 17 genera) bacterial specieslisted in Table 13. All bacterial species tested other than S.pneumoniae were negative except S. mitis and S. oralis. Ubiquity testswere performed using a collection of 98 S. pneumoniae strains includinghigh-level penicillin resistance (n=53), intermediate resistance (n=12)and sensitive (n=33) strains. There was a perfect correlation betweenPCR and standard susceptibility testing for 33 penicillin-sensitiveisolates. Among 12 S. pneumoniae isolates with intermediate penicillinresistance based on susceptibility testing, 11 had intermediateresistance based on PCR, but one S. pneumoniae isolate with penicillinMIC of 0.25 μg/ml showed a high-level penicillin resistance based ongenotyping. Among 53 isolates with high-level penicillin resistancebased on susceptibility testing, 51 had high-level penicillin resistancebased on PCR but two isolates with penicillin MIC>1 μg/ml showed anintermediate penicillin resistance based on genotyping. In general,there was a good correlation between the genotype and classical culturemethod for bacterial identification and susceptibility testing.

The sensitivity of the S. pneumoniae-specific assay with 40-cycle PCRprotocols was determined by using purified genomic DNA from 9 isolatesof S. pneumoniae. The detection limit was around 10 copies of genomicDNA for all of them.

Post-PCR Hybridization with Internal Probes.

The specificity of the multiplex PCR assay coupled with capture-probehybridization was tested with 98 strains of S. pneumoniae, 16 strains ofS. mitis and 3 strains of S. oralis. The internal probe specific to S.pneumoniae (SEQ ID NO. 1180) detected all 98 S. pneunoniae strains butdid not hybridize to the S. mitis and S. oralis amplicons. The fiveinternal probes specific to the high-level resistance amplicon (SEQ IDNOs. 1197, 1217-1220) detected all amplification patterns correspondingto high-level resistance. The two S. pneumoniae strains with penicillinMIC>1 μg/ml that showed an intermediate penicillin resistance based onPCR amplification were also intermediate resistance based on probehybridization. Similarly, among 12 strains with intermediate-penicillinresistance based on susceptibility testing, 11 showedintermediate-penicillin resistance based on hybridization with the fiveinternal probes specific to the intermediate and high-level resistanceamplicon (SEQ ID NOs. 1094, 1192-1193, 1214 and 1216). The straindescribed above having a penicillin MIC of 0.25 μg/ml which washigh-level penicillin resistance based on PCR amplification was alsohigh-level resistance based on probe hybridization. In summary, thecombination of the multiplex PCR and hybridization assays results in ahighly specific test for the detection of penicillin-resistantStreptococcus pneumoniae.

Assay II:

Bacterial Strains.

The specificity of the multiplex PCR assay was verified by using thesame strains as those used for the development of Assay I. Thepenicillin MICs (minimal inhibitory concentrations) were measured by thebroth dilution method according to the recommended protocol of NCCLS.

PCR Primers and Internal Probes.

The analysis of pbp1a sequences from S. pneumoniae strains with variouslevels of penicillin resistance from public databases and from thedatabase described in Example 18 allowed the design of two primerslocated in the constant region of pbp1a. PCR primer pair (SEQ ID NOs.2015 and 2016) was designed to amplify a 888-bp variable region of pbp1afrom all S. pneumoniae strains. A series of internal probes weredesigned for identification of the pbp1a mutations associated withpenicillin resistance in S. pneumoniae. For detection of high-levelpenicillin resistance (MICs≥1 μg/ml), three internal probes weredesigned (SEQ ID NOs. 2017-2019). Alternatively, ten other internalprobes were designed that can also be used for detection of high-levelresistance within the 888-bp pbp1a amplicon: (1) three internal probesfor identification of the amino acid substitutions Thr-371 to Ser or Alawithin the motif S370TMK (SEQ ID NOs. 2031-2033); (2) two internalprobes for detection of the amino acid substitutions Ile-459 to Met andSer-462 to Ala near the motif S428RN (SEQ ID NOs. 1135 and 2026); (3)two internal probes for identification of the amino acid substitutionsAsn-443 to Asp (SEQ ID NOs. 1134 and 2027); and (4) three internalprobes for detection of all sequence variations within another region(SEQ ID NOs. 2028-2030). For detection of high-level and intermediatepenicillin resistance (MICs≥0.25 μg/ml), four internal probes weredesigned (SEQ ID NOs. 2020-2023). Alternatively, six other internalprobes were designed for detection of the four consecutive amino acidsubstitutions T574SQF to A574TGY near the motif K557TG (SEQ ID NOs.2034-2039) that can also be used for detection of intermediate- andhigh-level resistance within the 888-bp pbp1a amplicon.

PCR Amplification.

For all bacterial species, amplification was performed from purifiedgenomic DNA using a PTC-200 thermocycler (MJ Research). 1 μl of genomicDNA at 0.1 ng/μ1, or 1 μl of a bacterial lysate, was transferred to a 19μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl(pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.08 μM (each) of the S.pneumoniae-specific primers SEQ ID NO. 1179 and SEQ ID NO. 1181, 0.4 μMof the pbp1a-specific primer SEQ ID NO. 2015, 1.2 μM of pbp1a-specificprimer SEQ ID NO. 2016, 0.05 mM bovine serum albumin (BSA), and 0.5 UTaq polymerase (Promega) coupled with TaqStart™ antibody. In order togenerate Digoxigenin (DIG)-labeled amplicons for capture probehybridization, 0.1×PCR DIG labeling four deoxynucleoside triphosphatesmix (Boehringer Mannheim GmbH) was used for amplification.

For determination of the sensitivities of the PCR assays, 10-folddilutions of purified genomic DNA were used to determine the minimalnumber of genome copies which can be detected.

Capture Probe Hybridization.

The DIG-labeled amplicons were hybridized to the capture probes bound to96-well plates as described for Assay I.

Results

Amplifications with the Multiplex PCR Assay.

The specificity of the assay was assessed by performing 40-cycle PCRamplifications with the panel of gram-positive (67 species from 12genera) and gram-negative (33 species from 17 genera) bacterial specieslisted in Table 13. All bacterial species tested other than S.pneumoniae were negative except S. mitis and S. oralis. Ubiquity testswere performed using a collection of 98 S. pneumoniae strains includinghigh-level penicillin resistance (n=53), intermediate resistance (n=12)and sensitive (n=33) strains. All the above S. pneumoniae strainsproduced the 888-bp amplicon corresponding to pbp1a and the 241-bpfragment corresponding to hexA.

The sensitivity of the S. pneumoniae-specific assay with 40-cycle PCRprotocols was determined by using purified genomic DNA from 9 isolatesof S. pneumoniae. The detection limit was around 10 copies of genomicDNA for all of them.

Post-PCR Hybridization with Internal Probes.

The specificity of the multiplex PCR assay coupled with capture-probehybridization was tested with 98 strains of S. pneumoniae, 16 strains ofS. mitis and 3 strains of S. oralis. The internal probe specific to S.pneumoniae (SEQ ID NO. 1180) detected all 98 S. pneunoniae strains butdid not hybridize to the S. mitis and S. oralis amplicons. The threeinternal probes (SEQ ID NOs 2017-2019) specific to high-level resistancedetected all the 43 strains with high-level penicillin resistance basedon susceptibility testing. Among 12 isolates withintermediate-penicillin resistance based on susceptibility testing, 11showed intermediate-penicillin resistance based on hybridization with 4internal probes (SEQ ID NOs. 2020-2023) and one strain having penicillinMIC of 0.25 μg/ml was misclassified as high-level penicillin resistance.In summary, the combination of the multiplex PCR and hybridizationassays results in a highly specific test for the detection ofpenicillin-resistant Streptococcus pneumoniae.

Example 21

Sequencing of the Vancomycin Resistance vanA, vanC1, vanC2 and vanC3Genes.

The publicly available sequences of the vanH-vanA-vanX-vanY locus oftransposon Tn1546 from E. faecalis, vanC1 sequence from one strain of E.gallinarum, vanC2 and vanC3 sequences from a variety of E. casseliflavusand E. flavescens strains, respectively, allowed the design of PCRprimers able to amplify the vanA, vanC1, vanC2 and vanC3 sequences ofseveral Enterococcus species. Using primer pairs van6877 and van9106(SEQ ID NOs. 1150 and 1155), vanC1-122 and vanC1-1315 (SEQ ID NOs. 1110and 1109), and vanC2C3-1 and vanC2C3-1064 (SEQ ID NOs. 1108 and 1107),it was possible to amplify and determine vanA sequences SEQ ID NOs.1049-1057, vanC1 sequences SEQ ID NOs. 1058-1059, vanC2 sequences SEQ IDNOs. 1060-1063 and vanC3 sequences SEQ ID NOs. 1064-1066, respectively.Four other PCR primers (SEQ ID NOs. 1151-1154) were also designed andused to complete the sequencing of vanA amplification products.

Example 22

Development of a PCR Assay for the Detection and Identification ofEnterococci at Genus and Species Levels and its Associated ResistanceGenes vanA and vanB.

The comparison of vanA and vanB sequences revealed conserved regionsallowing the design of PCR primers specific to both vanA and vanBsequences (Table 76). The PCR primer pair vanAB459 and vanAB830R (SEQ IDNOs. 1112 and 1111) was used in multiplex with the Enterococcus-specificprimers Encg313dF and Encg599c (SEQ ID NOs. 1137 and 1136) described inExample 11. Sequence alignment analysis of vanA and vanB sequencesrevealed regions suitable for the design of internal probes specific tovanA (SEQ ID NO. 1170) and vanB (SEQ ID NO. 1171). PCR amplification andagarose gel electrophoresis of the amplified products were performed asdescribed in Example 11. The optimal cycling conditions for maximumsensitivity and specificity were found to be 3 min. at 94° C., followedby forty cycles of two steps consisting of 1 second at 95° C. and 30seconds at 62° C., plus a terminal extension at 72° C. for 2 minutes.The specificity of the multiplex assay with 40-cycle PCR was verified byusing 0.1 nanogram of purified genomic DNA from a panel of bacterialisted in Table 10. The sensitivity of the multiplex assay with 40-cyclePCR was verified with three strains of E. casseliflavus, eight strainsof E. gallinarum, two strains of E. flavescens, two vancomycin-resistantstrains of E. faecalis and one vancomycin-sensitive strain of E.faecalis, three vancomycin-resistant strains of E. faecium, onevancomycin-sensitive strain of E. faecium and one strain of each of theother enterococcal species listed in Table 10. The detection limit was 1to 10 copies of genomic DNA, depending on the enterococcal speciestested. The vanA- and vanB-specific internal probes (SEQ ID NOs. 1170and 1171), as well as the E. faecalis- and E. faecium-specific internalprobes (SEQ ID NOs. 1174 and 602) and the internal probe specific to thegroup including E. casseliflavus, E. gallinarum and E. flavescens (SEQID NO. 1122) described in Example 11, were able to recognizevancomycin-resistant enterococcal species with high sensitivity,specificity and ubiquity showing a perfect correlation between thegenotypic and phenotypic analysis.

The format of the assay is not limited to the one described above. Aperson skilled in the art could adapt the assay for different formatssuch as PCR with real-time detection using molecular beacon probes.Molecular beacon probes designed to be used in this assay include, butare not limited to, SEQ ID NO. 1236 for the detection of E. faecalis,SEQ ID NO. 1235 for the detection of E. faecium, SEQ ID NO. 1240 for thedetection of vanA, and SEQ ID NO. 1241 for the detection of vanB.

Example 23

Development of a Multiplex PCR Assay for Detection and Identification ofVancomycin-Resistant Organisms Including Enterococcus faecalis,Enterococcus Faecium and the Group Including Enterococcus gallinarum,Enterococcus Casseliflavus, and Enterococcus flavescens.

The vanA and vanB genes encode the major glycopeptide resistancephenotypes in vancomycin resistant microorganisms. To design an assay todetect vancomycin resistant microorganisms, the nucleotide sequence ofthe vanA and vanB genes were analyzed. FIGS. 13 and 14 show a nucleotidesequence alignment of the vanA and vanB genes from the bacterial strainslisted in Tables 26 and 27, respectively. Shown above the sequencealignments is a consensus DNA sequence derived from the multiplesequences. The analysis of vanA and vanB sequences revealed conservedregions allowing design of PCR primer pairs (SEQ ID NOs. 1089 and 1090and SEQ ID NOs: 1090 and 1091) specific to vanA sequences (Table 66) andPCR primer pairs (SEQ ID NOs. 1095 and 1096 and SEQ ID NOs: 2298 and1096) specific to vanB sequences (Table 67). Shown below the sequencealignments in FIGS. 13 and 14 is the sequence and location of the vanAprimers SEQ ID NO: 1090 and 1091 and the vanB primers SEQ ID NOs: 2298and 1096, respectively. Also shown are the sequence and location ofmolecular beacon probes, SEQ ID NOs: 2299, 2300, designed for thedetection of the vanA and vanB amplification products, respectively. SEQID NO: 2299. The vanA molecular beacon (SEQ ID NO: 2299) contains a 5′carboxyfluorescein (FAM) flourophore. The vanB molecular beacon (SEQ IDNO: 2300) contains a sulforhodamine active ester (Texas Red) at its 5′end. Both beacons contain the nonfluorescent quencher moiety dabcylchloride (DABCYL) at their 3′ ends.

An internal control DNA, pERVd (SEQ ID NO: 2302) was designed for thevanR assay. The primers of SEQ ID NO: 1090 and SEQ ID NO: 1096 canhybridize to produce and amplification product from the internal controlDNA. A molecular beacon probe (SEQ ID NO: 2301) was designed to detectthe amplification product of the internal control DNA. SEQ ID NO: 2301contains a 5′ tetrachlorofluorescein (TET) fluorophore and the DABCYLquencher moiety at its 3′ end.

In a first experiment, the vanA-specific PCR primer pair (SEQ ID NOs.1089 and 1090) was used in multiplex with the vanB-specific PCR primerpair described in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 1095and 1096 in the present patent and SEQ ID NOs. 231 and 232 in the saidpatent). The comparison of vanC1, vanC2 and vanC3 sequences revealedconserved regions allowing design of PCR primers (SEQ ID NOs. 1101 and1102) able to generate a 158-bp amplicon specific to the group includingE. gallinarum, E. casseliflavus and E. flavescens (Table 68). ThevanC-specific PCR primer pair (SEQ ID NOs. 1101 and 1102) was used inmultiplex with the E. faecalis-specific PCR primer pair described in ourassigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 40 and 41 in the saidpatent) and with the E. faecium-specific PCR primer pair described inour patent publication WO98/20157 (SEQ ID NOs. 1 and 2 in the saidpublication). For both multiplexes, the optimal cycling conditions formaximum sensitivity and specificity were found to be 3 min. at 94° C.,followed by forty cycles of two steps consisting of 1 second at 95° C.and 30 seconds at 58° C., plus a terminal extension at 72° C. for 2minutes. Detection of the PCR products was made by electrophoresis inagarose gels (2%) containing 0.25 μg/ml of ethidium bromide. ThevanA-specific PCR primer pair (SEQ ID NOs. 1089 and 1090), thevanB-specific primer pair (SEQ ID NOs. 1095 and 1096) and thevanC-specific primer pair (SEQ ID NOs. 1101 and 1102) were tested fortheir specificity by using 0.1 nanogram of purified genomic DNA from apanel of 5 vancomycin-sensitive Enterococcus species, 3vancomycin-resistant Enterococcus species, 13 other gram-positivebacteria and one gram-negative bacterium. Specificity tests wereperformed with the E. faecium-specific PCR primer pair described in ourpatent publication WO98/20157 (SEQ ID NOs. 1 and 2 in the saidpublication) and with the E. faecalis-specific PCR primer pair describedin our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 40 and 41 in thesaid patent) on a panel of 37 gram-positive bacterial species. AllEnterococcus strains were amplified with high specificity showing aperfect correlation between the genotypic and phenotypic analysis. Thesensitivity of the assays was determined for several strains of E.gallinarum, E. casseliflavus, E. flavescens and vancomycin-resistant E.faecalis and E. faecium. Using each of the E. faecalis- and E.faecium-specific PCR primer pairs as well as vanA-, vanB- andvanC-specific PCR primers used alone or in multiplex as described above,the sensitivity ranged from 1 to 10 copies of genomic DNA.

The format of the assay is not limited to the one described above. Aperson skilled in the art could adapt the assay for different formatssuch as PCR with real-time detection using molecular beacon probes.Molecular beacon probes designed to be used in this assay include, butare not limited to, SEQ ID NO. 1238 for the detection of E. faecalis,SEQ ID NO. 1237 for the detection of E. faecium, SEQ ID NO. 1239 for thedetection of vanA, and SEQ ID NO. 1241 for the detection of vanB.

Another PCR assay was developed for the detection ofvancomycin-resistant E. faecium and vancomycin-resistant E. faecalis.This assay included two multiplex: (1) the first multiplex contained thevanA-specific primer pair (SEQ ID NOs. 1090-1091) and the vanB-specificPCR primer pair described in our assigned U.S. Pat. No. 5,994,066 (SEQID NOs. 1095 and 1096 in the present patent and SEQ ID NOs. 231 and 232in the said patent), and (2) the second multiplex contained the E.faecalis-specific PCR primer pair described in our assigned U.S. Pat.No. 5,994,066 (SEQ ID NOs. 40 and 41 in the said patent) and the E.faecium-specific PCR primer pair described in our patent publicationWO98/20157 (SEQ ID NOs. 1 and 2 in the said publication). For bothmultiplexes, the optimal cycling conditions for maximum sensitivity andspecificity were found to be 3 min. at 94° C., followed by forty cyclesof two steps consisting of 1 second at 95° C. and 30 seconds at 58° C.,plus a terminal extension at 72° C. for 2 minutes. Detection of the PCRproducts was made by electrophoresis in agarose gels (2%) containing0.25 μg/ml of ethidium bromide. The two multiplexes were tested fortheir specificity by using 0.1 nanogram of purified genomic DNA from apanel of two vancomycin-sensitive E. faecalis strains, twovancomycin-resistant E. faecalis strains, two vancomycin-sensitive E.faecium strains, two vancomycin-resistant E. faecium strains, 16 otherenterococcal species and 31 other gram-positive bacterial species. Allthe E. faecium and E. faecalis strains were amplified with highspecificity showing a perfect correlation between the genotypic analysisand the susceptibility to glycopeptide antibiotics (vancomycin andteicoplanin). The sensitivity of the assay was determined for twovancomycin-resistant E. faecalis strains and two vancomycin-resistant E.faecium strains. The detection limit was 5 copies of genomic DNA for allthe strains.

This multiplex PCR assay was coupled with capture-probe hybridization.Four internal probes were designed: one specific to the vanA amplicon(SEQ ID NO. 2292), one specific to the vanB amplicon (SEQ ID NO. 2294),one specific to the E. faecalis amplicon (SEQ ID NO. 2291) and onespecific to the E. faecium amplicon (SEQ ID NO. 2287). Each of theinternal probes detected their specific amplicons with high specificityand sensitivity.

The VanR Assay

Development

In a next set of experiments, a multiplex real-time PCR reaction (theVanR assay) using the vanA primer pair SEQ ID NO: 1090 and 1091, thevanB primer pair SEQ ID NO: 1096 and 2298, the internal control DNA SEQID NO: 2302, and the molecular beacon probes SEQ ID NOs: 2299, 2300, and2301 was performed.

For the template DNA, purified genomic DNA from vanA resistantEnterococcus faecium or vanB resistant Enterococcus faecalis wasobtained from strains grown on blood agar plates under standardconditions. Genomic DNA was extracted from the cultures using the GNOMEkit (QBIOgene, Carlsbad, Calif.) according to the manufacturer'sinstructions. Template DNA samples were treated with RNase prior toquanatitation. Genomic DNA concentration was determined and the qualityof the genomic DNA preparations was confirmed using conventionalmethods.

Lyophilized reagents listed in Table 24 were used for the VanR assay.The lyophilized reagents were reconstituted with 225 μl diluent (116 mMTris-HCl, pH 8.3, 11.6 mM KCl, 3.48 mM MgCl₂, 5.8 mM NH₂SO₄) andsubsequently divided into 25 μl aliquots. 0.5, 2.5, 5, 10 or 20 copiesof template DNA was added to each of 5 replicate reactions.

The VanR assay PCR was run in a SMART CYCLER™ PCR machine under thefollowing conditions: 60° C. for 6 sec, followed by 95° C. for 900 sec,followed by 45 cycles of 95° C. for 5 seconds, 63° C. for 10 sec and 72°C. for 20 sec. The fluorescent readout from the FAM channel (vanA) andthe Texas Red channel (vanB) of the reactions is shown in FIGS. 13A and13B, respectively. The VanR assay is capable of detecting 5 copies ofvanA target DNA in a sample (FIG. 13A) and of detecting 10 copies ofvanB target DNA in a sample (FIG. 13B). For each concentration oftemplate DNA, the number of positive assay results out of each of thefive replicates was recorded (Table 15).

Specificity

To demonstrate the specificity of the VanR assay, vanC, vanD, vanE, andvanG resistant enterococci, other closely related bacteria, normal andpathogenic anal or fecal flora, or human DNA as listed in Tables 16-18were assayed as follows: VanR assay lyophilized reagents (Table 38) werereconstituted with 225 μl diluent and aliquoted as described above.Template DNA was prepared using conventional methods and diluted insample preparation buffer to a final concentration of 0.33 ng/μl. 3 μltemplate DNA was added to each master mix, and the reaction was carriedout as described above in the SMART CYCLER™ PCR machine. 3 μlelectrophoresis loading buffer was added to the reactions uponcompletion, and 15 μl of each reaction was run on an agarose gel to viewthe PCR amplification products, as shown in FIGS. 14-17.

To assess the specificity of the molecular beacons in the VanR assay,PCR assays were preformed to test cross-reactivity with amplified vanAand vanB target DNA and internal control DNA. The reaction componentslisted in Table 34 were each added to premix in Table 33. For eachreaction, 25.8 μl of the final mix was combined with 3 μl template DNA(either 25 copies vanA DNA or 50 copies vanB DNA) or TE buffer (1×), asindicated. The reactions were performed in the SMART CYCLER™ PCR machineusing the VanR assay conditions as described above. Table 35 shows theresults of the experiment. Positive results were obtained in the FAMchannel in every reaction that contained vanA template DNA and the vanAprimers. Positive results were obtained in the Texas Red channel inevery reaction that contained vanB template DNA and vanB primers.Positive results obtained in the TET channel in every reaction thatcontained internal control template DNA and internal control primers.

Negative results were obtained in each channel for every reaction thatdid not contain a target DNA that could be amplified and detected usingthe indicated molecular beacon probe. In other words, the molecularbeacon probes did not cross-react with non-specific DNAs. FIGS. 18A-20Bshow the fluorescent readouts of the experiments.

Validation with Clinical Samples

To validate the VanR assay, the sensitivity of the VanR assay wasperformed on clinical samples. Briefly, the VanR assay lyophilizedreagents listed in Table 38 were reconstituted in 225 μl diluent andaliquoted as described above. DNA isolation and quantitation wasperformed using conventional techniques. The samples were processed in aSMART CYCLER™ PCR machine using the VanR reaction conditions describedabove.

FIG. 21 shows the fluorescent readout in the FAM (vanA) channel and theTexas Red channel (vanB) from a specimen that is vanA resistant. Thereaction gave a positive result in the FAM channel but not the Texas Redchannel. FIG. 22 shows the fluorescent readaout in the FAM and Texas Redchannels from a combination of a vanA resistant specimen and a vanBresistant specimen. Positive results were obtained in both the FAM andthe Texas Red channel. FIG. 23 shows the fluorescent readout in the FAMand Texas Red channels from a vanB positive specimen. The results werenegative in the FAM channel, and positive in the Texas Red channel.

Sensitivity

To assess the sensitivity of the VanR assay, template DNA isolated fromenterococci from various geographic regions, and on template DNA fromvanB resistant bacterial species other than enterococci was isolated andtested in the VanR assay as described above. Table 36 lists theenterococcal strains from various regions in the world tested in theassay. The table identifies the vanA and vanB phenotype of each strain.PCR amplification products were detected by running a sample of eachreaction on an agarose gel, shown in FIG. 24. vanA or vanB PCRamplification products were detectable in each of the strains tested.Table 37 lists non-enterococcal, vanB resistant strains tested in theVanR assay. The PCR amplification products are shown in FIG. 25. Thereaction produced an amplification product of the expected size for thevanB amplicon.

These results demonstrate that the VanR assay is sensitive and specific.

Example 24

Universal Amplification Involving the EF-G (fusA) Subdivision of tufSequences.

As shown in FIG. 3, primers SEQ ID NOs. 1228 and 1229 were designed toamplify the region between the end of fusA and the beginning of tufgenes in the str operon. Genomic DNAs from a panel of 35 strains weretested for PCR amplification with those primers. In the initialexperiment, the following strains showed a positive result: Abiotrophiaadiacens ATCC 49175, Abiotrophia defectiva ATCC 49176, Bacillus subtilisATCC 27370, Closridium difficile ATCC 9689, Enterococcus avium ATCC14025, Enterococcus casseliflavus ATCC 25788, Enterococcus cecorum ATCC43198, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC19434, Enterococcus flavescens ATCC 49996, Enterococcus gallinarum ATCC49573, Enterococcus solitarius ATCC 49428, Escherichia coli ATCC 11775,Haemophilus influenzae ATCC 9006, Lactobacillus acidophilus ATCC 4356,Peptococcus niger ATCC 27731, Proteus mirabilis ATCC 25933,Staphylococcus aureus ATCC 43300, Staphylococcus auricularis ATCC 33753,Staphylococcus capitis ATCC 27840, Staphylococcus epidemidis ATCC 14990,Staphylococcus haemolyticus ATCC 29970, Staphylococcus hominis ATCC27844, Staphylococcus lugdunensis ATCC 43809, Staphylococcussaprophyticus ATCC 15305, Staphylococcus simulans ATCC 27848, andStaphylococcus warneri ATCC 27836. This primer pair could amplifyadditional bacterial species; however, there was no amplification forsome species, suggesting that the PCR cycling conditions could beoptimized or the primers modified. For example, SEQ ID NO. 1227 wasdesigned to amplify a broader range of species.

In addition to other possible primer combinations to amplify the regioncovering fusA and tuf, FIG. 3 illustrates the positions of amplificationprimers SEQ ID NOs. 1221-1227 which could be used for universalamplification of fusA segments. All of the above mentioned primers (SEQID NOs. 1221-1229) could be useful for the universal and/or the specificdetection of bacteria.

Moreover, different combinations of primers SEQ ID NOs. 1221-1229,sometimes in combination with tuf sequencing primer SEQ ID NO. 697, wereused to sequence portions of the str operon, including the intergenicregion. In this manner, the following sequences were generated: SEQ IDNOs. 1518-1526, 1578-1580, 1786-1821, 1822-1834, 1838-1843, 2184, 2187,2188, 2214-2249, and 2255-2269.

Example 25

DNA Fragment Isolation from Staphylococcus saprophyticus by ArbitrarilyPrimed PCR.

DNA sequences of unknown coding potential for the species-specificdetection and identification of Staphylococcus saprophyticus wereobtained by the method of arbitrarily primed PCR (AP-PCR).

AP-PCR is a method which can be used to generate specific DNA probes formicroorganisms (Fani et al., 1993, Molecular Ecology 2:243-250). Adescription of the AP-PCR protocol used to isolate a species-specificgenomic DNA fragment from Staphylococcus saprophyticus follows. Twentydifferent oligonucleotide primers of 10 nucleotides in length (allincluded in the AP-PCR kit OPAD (Operon Technologies, Inc., Alameda,Calif.)) were tested systematically with DNAs from 5 bacterial strainsof Staphylococcus saprophyticus as well as with bacterial strains of 27other staphylococcal (non-S. saprophyticus) species. For all bacterialspecies, amplification was performed directly from one μL (0.1 ng/μL) ofpurified genomic DNA. The 25 μL PCR reaction mixture contained 50 mMKCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 1.2 μM ofonly one of the 20 different AP-PCR primers OPAD, 200 μM of each of thefour dNTPs, 0.5 U of Taq DNA polymerase (Promega Corp., Madison, Wis.)coupled with TaqStart™ antibody (Clontech Laboratories Inc., Palo Alto,Calif.). PCR reactions were subjected to cycling using a MJ ResearchPTC-200 thermal cycler as follows: 3 min at 96° C. followed by 42 cyclesof 1 min at 94° C. for the denaturation step, 1 min at 31° C. for theannealing step and 2 min at 72° C. for the extension step. A finalextension step of 7 min at 72° C. followed the 42 cycles to ensurecomplete extension of PCR products. Subsequently, twenty microliters ofthe PCR-amplified mixture were resolved by electrophoresis on a 1.5%agarose gel containing 0.25 μg/ml of ethidium bromide. The size of theamplification products was estimated by comparison with a 50-bpmolecular weight ladder.

Amplification patterns specific for Staphylococcus saprophyticus wereobserved with the AP-PCR primer OPAD-16 (sequence: 5′-AACGGGCGTC-3′).Amplification with this primer consistently showed a band correspondingto a DNA fragment of approximately 380 bp for all Staphylococcussaprophyticus strains tested but not for any of the other staphylococcalspecies tested.

The band corresponding to the 380 bp amplicon, specific and ubiquitousfor S. saprophyticus based on AP-PCR, was excised from the agarose geland purified using the QIAquick gel extraction kit (QIAGEN Inc.). Thegel-purified DNA fragment was cloned into the T/A cloning site of thepCR 2.1™ plasmid vector (Invitrogen Inc.) using T4 DNA ligase (NewEngland BioLabs). Recombinant plasmids were transformed into E. coliDH5α competent cells using standard procedures. All reactions wereperformed according to the manufacturer's instructions. Plasmid DNAisolation was done by the method of Birnboim and Doly (Nucleic AcidRes., 1979, 7:1513-1523) for small-scale preparations. All plasmid DNApreparations were digested with the EcoRI restriction endonuclease toensure the presence of the approximately 380 bp AP-PCR insert into theplasmid. Subsequently, a large-scale and highly purified plasmid DNApreparation was performed from two selected clones shown to carry theAP-PCR insert by using the QIAGEN plasmid purification kit (midiformat). These large-scale plasmid preparations were used for automatedDNA sequencing.

The 380 bp nucleotide sequence was determined for three strains of S.saprophyticus (SEQ ID NOs. 74, 1093, and 1198). Both strands of theAP-PCR insert from the two selected clones were sequenced by thedideoxynucleotide chain termination sequencing method with SP6 and T7sequencing primers by using the Applied Biosystems automated DNAsequencer (model 373A) with their PRISM™ Sequenase® TerminatorDouble-stranded DNA Sequencing Kit (Applied Biosystems, Foster City,Calif.).

Optimal species-specific amplification primers (SEQ ID NOs. 1208 and1209) have been selected from the sequenced AP-PCR Staphylococcussaprophyticus DNA fragments with the help of the primer analysissoftware Oligo™ 5.0 (National BioSciences Inc.). The selected primerswere tested in PCR assays to verify their specificity and ubiquity. Dataobtained with DNA preparations from reference ATCC strains of 49gram-positive and 31 gram-negative bacterial species, including 28different staphylococcal species, indicate that the selected primerpairs are specific for Staphylococcus saprophyticus since noamplification signal has been observed with DNAs from the otherstaphylococcal or bacterial species tested. This assay was able toamplify efficiently DNA from all 60 strains of S. saprophyticus fromvarious origins tested. The sensitivity level achieved for three S.saprophyticus reference ATCC strains was around 6 genome copies.

Example 26

Sequencing of Prokaryotic tuf Gene Fragments.

The comparison of publicly available tuf sequences from a variety ofbacterial species revealed conserved regions, allowing the design of PCRprimers able to amplify tuf sequences from a wide range of bacterialspecies. Using primer pair SEQ ID NOs. 664 and 697, it was possible toamplify and determine tuf sequences SEQ ID NOs.: 1-73, 75-241, 607-618,621, 662, 675, 717-736, 868-888, 932, 967-989, 992, 1002, 1572-1575,1662-1663, 1715-1733, 1835-1837, 1877-1878, 1880-1881, 2183, 2185, 2200,2201, and 2270-2272.

Example 27

Sequencing of Procaryotic recA Gene Fragments.

The comparison of publicly available recA sequences from a variety ofbacterial species revealed conserved regions, allowing the design of PCRprimers able to amplify recA sequences from a wide range of bacterialspecies. Using primer pairs SEQ ID NOs. 921-922 and 1605-1606, it waspossible to amplify and determine recA sequences SEQ ID NOs.: 990-991,1003, 1288-1289, 1714, 1756-1763, 1866-1873 and 2202-2212.

Example 28

Specific Detection and Identification of Escherichia coli/Shigella sp.Using tuf Sequences.

The analysis of tuf sequences from a variety of bacterial speciesallowed the selection of PCR primers (SEQ ID NOs. 1661 and 1665) and ofan internal probe (SEQ ID NO. 2168) specific to Escherichiacoli/Shigella sp. The strategy used to design the PCR primers was basedon the analysis of a multiple sequence alignment of various tufsequences. The multiple sequence alignment included the tuf sequences ofEscherichia coli/Shigella sp. as well as tuf sequences from otherspecies and bacterial genera, especially representatives of closelyrelated species. A careful analysis of this alignment allowed theselection of oligonucleotide sequences which are conserved within thetarget species but which discriminate sequences from other species,especially from the closely related species, thereby permitting thespecies-specific and ubiquitous detection and identification of thetarget bacterial species.

The chosen primer pair, oligos SEQ ID NOs. 1661 and 1665, gives anamplification product of 219 bp. Standard PCR was carried out using 0.4μM of each primer, 2.5 mM MgCl₂, BSA 0.05 mM, 50 mM KCl, 10 mM Tris-HCl(pH 9.0), 0.1% Triton X-100, dNTPs 0.2 mM (Pharmacia), 0.5 U Taq DNApolymerase (Promega) coupled with TaqStart™ antibody (ClontechLaboratories Inc.), 1 μl of genomic DNA sample in a final volume of 20μl using a PTC-200 thermocycler (MJ Research). The optimal cyclingconditions for maximum sensitivity and specificity were 3 minutes at 95°C. for initial denaturation, then forty cycles of two steps consistingof 1 second at 95° C. and 30 seconds at 60° C., followed by terminalextension at 72° C. for 2 minutes. Detection of the PCR products wasmade by electrophoresis in agarose gels (2%) containing 0.25 μg/ml ofethidium bromide. Visualization of the PCR products was made under UV at254 nm.

Specificity of the assay was tested by adding to the PCR reactions 0.1ng of genomic DNA from each of the following bacterial species:Escherichia coli (7 strains), Shigella sonnei, Shigella flexneri,Shigella dysenteriae, Salmonella typhimyurium, Salmonella typhi,Salmonella enteritidis, Tatumella ptyseos, Klebsiella pneumoniae (2strains), Enterobacter aerogenes, Citrobacter farmeri, Campylobacterjejuni, Serratia marcescens. Amplification was observed only for theEscherichia coli and Shigella sp. strains listed and Escherichiafergusonii. The sensitivity of the assay with 40-cycle PCR was verifiedwith one strain of E. coli and three strains of Shigella sp. Thedetection limit for E. coli and Shigella sp. was 1 to 10 copies ofgenomic DNA, depending on the strains tested.

Example 29

Specific Detection and Identification of Klebsiella pneumoniae UsingatpD Sequences.

The analysis of atpD sequences from a variety of bacterial speciesallowed the selection of PCR primers specific to K. pneumoniae. Theprimer design strategy is similar to the strategy described in Example28 except that atpD sequences were used in the alignment.

Two K. pneumoniae-specific primers were selected, (SEQ ID NOs. 1331 and1332) which give an amplification product of 115 bp. Standard PCR wascarried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM of eachprimer as described in Example 28. The optimal cycling conditions formaximum sensitivity and specificity were as follow: three minutes at 95°C. for initial denaturation, then forty cycles of two steps consistingof 1 second at 95° C. and 30 seconds at 55° C., followed by terminalextension at 72° C. for 2 minutes.

Specificity of the assay was tested by adding to the PCR reactions 0.1ng of genomic DNA from each of the following bacterial species:Klebsiella pneumoniae (2 strains), Klebsiella ornitholytica, Klebsiellaoxytoca (2 strains), Klebsiella planticola, Klebsiella terrigena,Citrobacter freundii, Escherichia coli, Salmonella cholerasuis typhi,Serratia marcescens, Enterobacter aerogenes, Proteus vulgaris, Kluyveraascorbata, Kluyvera georgiana, Kluyvera cryocrescens and Yersiniaenterolitica. Amplification was detected for the two K. pneumoniaestrains, K. planticola, K. terrigena and the three Kluyvera speciestested. Analysis of the multiple alignment sequence of the atpD geneallowed the design of an internal probe SEQ ID NO. 2167 which candiscrimate Klebsiella pneumoniae from other Klebsiella sp. and Kluyverasp. The sensitivity of the assay with 40-cycle PCR was verified with onestrain of K. pneumoniae. The detection limit for K. pneumoniae wasaround 10 copies of genomic DNA.

Example 30

Specific Detection and Identification of Acinetobacter baumannii UsingatpD Sequences.

The analysis of atpD sequences from a variety of bacterial speciesallowed the selection of PCR primers specific to Acinetobacterbaumannii. The primer design strategy is similar to the strategydescribed in Example 28.

Two A. baumannii-specific primers were selected, SEQ ID NOs. 1690 and1691, which give an amplification product of 233 bp. Standard PCR wascarried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM of eachprimer as described in Example 28. The optimal cycling conditions formaximum sensitivity and specificity were as follow: three minutes at 95°C. for initial denaturation, then forty cycles of two steps consistingof 1 second at 95° C. and 30 seconds at 60° C., followed by terminalextension at 72° C. for 2 minutes.

Specificity of the assay was tested by adding to the PCR reactions 0.1ng of genomic DNA from each of the following bacterial species:Acinetobacter baumannii (3 strains), Acinetobacter anitratus,Acinetobacter lwoffi, Serratia marcescens, Enterobacter cloacae,Enterococcus faecalis, Pseudomonas aeruginosa, Psychrobacterphenylpyruvicus, Neisseria gonorrheoae, Haemophilus haemoliticus,Yersinia enterolitica, Proteus vulgaris, Eikenella corrodens,Escherichia coli. Amplification was detected only for A. baumannii, Aanitratus and A. lwoffi. The sensitivity of the assay with 40-cycle PCRwas verified with two strains of A. baumannii. The detection limit forthe two A. baumannii strains tested was 5 copies of genomic DNA.Analysis of the multiple alignment sequence of the atpD gene allowed thedesign of a A. baumannii-specific internal probe (SEQ ID NO. 2169).

Example 31

Specific Detection and Identification of Neisseria gonorrhoeae Using tufSequences.

The analysis of tuf sequences from a variety of bacterial speciesallowed the selection of PCR primers specific to Neisseria gonorrhoeae.The primer design strategy is similar to the strategy described inExample 28.

Two N. gonorrhoeae-specific primers were selected, SEQ ID NOs. 551 and552, which give an amplification product of 139 bp. PCR amplificationwas carried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM ofeach primer as described in Example 28. The optimal cycling conditionsfor maximum sensitivity and specificity were as follow: three minutes at95° C. for initial denaturation, then forty cycles of two stepsconsisting of 1 second at 95° C. and 30 seconds at 65° C., followed byterminal extension at 72° C. for 2 minutes.

Specificity of the assay was tested by adding into the PCR reactions,0.1 ng of genomic DNA from each of the following bacterial species:Neisseria gonorrhoeae (19 strains), Neisseria meningitidis (2 strains),Neisseria lactamica, Neisseria flavescens, Neisseria animalis, Neisseriacanis, Neisseria cuniculi, Neisseria elongata, Neisseria mucosa,Neisseria polysaccharea, Neisseria sicca, Neisseria subflava, Neisseriaweaveri. Amplification was detected only for N. gonorrhoeae, N. siccaand N. polysaccharea. The sensitivity of the assay with 40-cycle PCR wasverified with two strains of N. gonorrhoeae. The detection limit for theN. gonorrhoeae strains tested was 5 copies of genomic DNA. Analysis ofthe multiple alignment sequence of the tuf gene allowed the design of aninternal probe, SEQ ID NO. 2166, which can discriminate N. gonorrhoeaefrom N. sicca and N. polysaccharea.

Example 32

Sequencing of Bacterial gyrA and parC Gene Fragments. Sequencing ofBacterial gyrA and parC Fragments.

One of the major mechanism of resistance to quinolone in variousbacterial species is mediated by target changes (DNA gyrase and/ortopoisomerase IV). These enzymes control DNA topology and are vital forchromosome function and replication. Each of these enzymes is a tetramercomposed of two subunits: GyrA and GyrB forming A₂B₂ complex in DNAgyrase; and ParC and ParE forming and C₂E₂ complex in DNA topoisomeraseIV. It has been shown that they are hotspots, called thequinolone-resistance-determining region (QRDR) for mutations within gyrAthat encodes for the GyrA subunit of DNA gyrase and within parC thatencodes the parC subunit of topoisomerase IV.

In order to generate a database for gyrA and parC sequences that can beused for design of primers and/or probes for the specific detection ofquinolone resistance in various bacterial species, gyrA and parC DNAfragments selected from public database (GenBanK and EMBL) from avariety of bacterial species were used to design oligonucleotideprimers.

Using primer pair SEQ ID NOs. 1297 and 1298, it was possible to amplifyand determine gyrA sequences from Klebsiella oxytoca (SEQ ID NO. 1764),Klebsiella pneumoniae subsp. ozaneae (SEQ ID NO. 1765), Klebsiellaplanticola (SEQ ID NO. 1766), Klebsiella pneumoniae (SEQ ID NO. 1767),Klebsiella pneumoniae subsp. pneumoniae (two strains) (SEQ ID NOs.1768-1769), Klebsiella pneumoniae subsp. rhinoscleromatis (SEQ ID NO.1770), Klebsiella terrigena (SEQ ID NO. 1771), Kluyvera ascorbata (SEQID NO. 2013), Kluyvera georgiana (SEQ ID NO. 2014) and Escherichia coli(4 strains) (SEQ ID NOs. 2277-2280). Using primer pair SEQ ID NOs. 1291and 1292, it was possible to amplify and determine gyrA sequences fromLegionella pneumophila subsp. pneumophila (SEQ ID NO. 1772), Proteusmirabilis (SEQ ID NO. 1773), Providencia rettgeri (SEQ ID NO. 1774),Proteus vulgaris (SEQ ID NO. 1775) and Yersinia enterolitica (SEQ ID NO.1776). Using primer pair SEQ ID NOs. 1340 and 1341, it was possible toamplify and determine gyrA sequence from Staphylococcus aureus (SEQ IDNO. 1255).

Using primers SEQ ID NOs. 1318 and 1319, it was possible to amplify anddetermine parC sequences from K. oxytoca (two strains) (SEQ ID NOs.1777-1778), Klebsiella pneumoniae subsp. ozaenae (SEQ ID NO. 1779),Klebsiella planticola (SEQ ID NO. 1780), Klebsiella pneumoniae (SEQ IDNO. 1781), Klebsiella pneumoniae subsp. pneumoniae (two strains) (SEQ IDNOs. 1782-1783), Klebsiella pneumoniae subsp. rhinoscleromatis (SEQ IDNO. 1784) and Klebsiella terrigena (SEQ ID NO. 1785).

Example 33

Development of a PCR Assay for the Specific Detection and Identificationof Staphylococcus aureus and its Quinolone Resistance Genes gyrA andparC.

The analysis of gyrA and parC sequences from a variety of bacterialspecies revealed conserved regions allowing the design of PCR primersspecific to the quinolone-resistance-determining region (QRDR) of gyrAand parC from Staphylococcus aureus. PCR primer pair SEQ ID NOs. 1340and 1341 was designed to amplify the gyrA sequence of S. aureus, whereasPCR primer pair SEQ ID NOs. 1342 and 1343 was designed to amplify S.aureus parC. The comparison of gyrA and parC sequences from S. aureusstrains with various levels of quinolone resistance allowed theidentification of amino acid substitutions Ser-84 to Leu, Glu-88 to Glyor Lys in the GyrA subunit of DNA gyrase encoded by gyrA and amino acidchanges Ser-80 to Phe or Tyr and Ala-116 to Glu in the ParC subunit oftopoisomerase IV encoded by parC. These amino acid substitutions in GyrAand ParC subunits occur in isolates with intermediate- or high-levelquinolone resistance. Internal probes for the specific detection ofwild-type S. aureus gyrA (SEQ ID NO. 1940) and wild-type S. aureus parC(SEQ ID NO. 1941) as well as internal probes for the specific detectionof each of the gyrA (SEQ ID NOs. 1333-1335) and parC mutationsidentified in quinolone-resistant S. aureus (SEQ ID NOs. 1336-1339) weredesigned.

The gyrA- and parC-specific primer pairs (SEQ ID NOs. 1340-1341 and SEQID NOs. 1342-1343) were used in multiplex. PCR amplification was carriedout on PTC-200 thermocyclers (MJ Research) using 0.3, 0.3, 0.6 and 0.6μM of each primers, respectively, as described in Example 28. Theoptimal cycling conditions for maximum sensitivity and specificity were3 minutes at 95° C. for initial denaturation, then forty cycles of twosteps consisting of 1 second at 95° C. and 30 seconds at 62° C.,followed by terminal extension at 72° C. for 2 minutes. Detection of thePCR products was made by electrophoresis in agarose gels (2%) containing0.25 μg/ml of ethidium bromide. The specificity of the multiplex assaywith 40-cycle PCR was verified by using 0.1 ng of purified genomic DNAfrom a panel of gram-positive bacteria. The list included the following:Abiotrophia adiacens, Abiotrophia defectiva, Bacillus cereus, Bacillusmycoides, Enterococcus faecalis (2 strains), Enterococcus flavescens,Gemella morbillorum, Lactococcus lactis, Listeria innocua, Listeriamonocytogenes, Staphylococcus aureus (5 strains), Staphylococcusauricalis, Staphylococcus capitis subsp. urealyticus, Staphylococcuscarnosus, Staphylococcus chromogenes, Staphylococcus epidermidis (3strains), Staphylococcus gallinarum, Staphylococcus haemolyticus (2strains), Staphylococcus hominis, Staphylococcus hominis subsp hominis,Staphylococcuslentus, Staphylococcus lugdunensis, Staphylococcussaccharolyticus, Staphylococcus saprophyticus (3 strains),Staphylococcus simulans, Staphylococcus warneri, Staphylococcus xylosus,Streptococcus agalactiae, Streptococcus pneumoniae. Strong amplificationof both gyrA and parC genes was only detected for the S. aureus strainstested. The sensitivity of the multiplex assay with 40-cycle PCR wasverified with one quinolone-sensitive and four quinolone-resistantstrains of S. aureus. The detection limit was 2 to 10 copies of genomicDNA, depending on the strains tested.

Detection of the hybridization with the internal probes was performed asdescribed in Example 7. The internal probes specific to wild-type gyrAand parC of S. aureus and to the gyrA and parC variants of S. aureuswere able to recognize two quinolone-resistant and onequinolone-sensitive S. aureus strains showing a perfect correlation withthe susceptibility to quinolones.

The complete assay for the specific detection of S. aureus and itssusceptibility to quinolone contains the Staphylococcus-specific primers(SEQ ID NOs. 553 and 575) described in Example 7 and the multiplexcontaining the S. aureus gyrA- and parC-specific primer pairs (SEQ IDNOs. 1340-1341 and SEQ ID NOs. 1342-1343). Amplification is coupled withpost-PCR hybridization with the internal probe specific to S. aureus(SEQ ID NO. 587) described in Example 7 and the internal probes specificto wild-type S. aureus gyrA and parC (SEQ ID NOs. 1940-1941) and to theS. aureus gyrA and parC variants (SEQ ID NOs. 1333-1338).

An assay was also developed for the detection of quinolone-resistant S.aureus using the SmartCycler (Cepheid). Real-time detection is based onthe use of S. aureus parC-specific primers (SEQ ID NOs. 1342 and 1343)and the Staphylococcus-specific primers (SEQ ID NOs. 553 and 575)described in Example 7. Internal probes were designed for molecularbeacon detection of the wild-type S. aureus parC (SEQ ID NO.1939), fordetection of the Ser-80 to Tyr or Phe amino acid substitutions in theParC subunit encoded by S. aureus parC (SEQ ID NOs. 1938 and 1955) andfor detection of S. aureus (SEQ ID NO. 2282).

Example 34

Development of a PCR Assay for the Detection and Identification ofKlebsiella Pneumoniae and its Quinolone Resistance Genes gyrA and parC.

The analysis of gyrA and parC sequences from a variety of bacterialspecies from the public databases and from the database described inExample 32 revealed conserved regions allowing the design of PCR primersspecific to the quinolone-resistance-determining region (QRDR) of gyrAand parC from K. pneumoniae. PCR primer pair SEQ ID NOs. 1936 and 1937,or pair SEQ ID NOs. 1937 and 1942, were designed to amplify the gyrAsequence of K. pneumoniae, whereas PCR primer pair SEQ ID NOs. 1934 and1935 was designed to amplify K. pneumoniae parC sequence. An alternativepair, SEQ ID NOs. 1935 and 1936, can also amplify K. pneumoniae parC.The comparison of gyrA and parC sequences from K. pneumoniae strainswith various levels of quinolone resistance allowed the identificationof amino acid substitutions Ser-83 to Tyr or Phe and Asp-87 to Gly orAla and Asp-87 to Asn in the GyrA subunit of DNA gyrase encoded by gyrAand amino acid changes Ser-80 to Ile or Arg and Glu-84 to Gly or Lys inthe ParC subunit of topoisomerase IV encoded by parC. These amino acidsubstitutions in the GyrA and ParC subunits occur in isolates withintermediate- or high-level quinolone resistance. Internal probes forthe specific detection of wild-type K. pneumoniae gyrA (SEQ ID NO. 1943)and wild-type K. pneumoniae parC (SEQ ID NO. 1944) as well as internalprobes for the specific detection of each of the gyrA (SEQ ID NOs.1945-1949) and parC mutations identified in quinolone-resistant K.pneumoniae (SEQ ID NOs. 1950-1953) were designed.

Two multiplex using the K. pneumoniae gyrA- and parC-specific primerpairs were used: the first multiplex contained K. pneumoniaegyrA-specific primers (SEQ ID NOs. 1937 and 1942) and K. pneumoniaeparC-specific primers (SEQ ID NOs. 1934 and 1935) and the secondmultiplex contained K. pneumoniae gyrA/parC-specific primer (SEQ ID NOs.1936), K. pneumoniae gyrA-specific primer (SEQ ID NO. 1937) and K.pneumoniae parC-specific primer (SEQ ID NO. 1935). Standard PCR wascarried out on PTC-200 thermocyclers (MJ Research) using for the firstmultiplex 0.6, 0.6, 0.4, 0.4 μM of each primer, respectively, and forthe second multiplex 0.8, 0.4, 0.4 μM of each primer, respectively. PCRamplification and agarose gel electrophoresis of the amplified productswere performed as described in Example 28. The optimal cyclingconditions for maximum sensitivity and specificity were 3 minutes at 95°C. for initial denaturation, then forty cycles of two steps consistingof 1 second at 95° C. and 30 seconds at 62° C., followed by terminalextension at 72° C. for 2 minutes. The specificity of the two multiplexassays with 40-cycle PCR was verified by using 0.1 ng of purifiedgenomic DNA from a panel of gram-negative bacteria. The list included:Acinetobacter baumannii, Citrobacter freundii, Eikenella corrodens,Enterobacter aerogenes, Enterobacter cancerogenes, Enterobacter cloacae,Escherichia coli (10 strains), Haemophilus influenzae, Klebsiellapneumoniae, Klebsiella ornitholytica, Klebsiella oxytoca (2 strains),Klebsiella planticola, Klebsiella terrigena, Kluyvera ascorbata,Kluyvera cryocrescens, Kluyvera georgiana, Neisseria gonorrhoeae,Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonellacholeraesuis subsp. typhimurium, Salmonella enteritidis, Serratialiquefaciens, Serratia marcescens and Yersinia enterocolytica. For bothmultiplex, strong amplification of both gyrA and parC was observed onlyfor the K. pneumoniae strain tested. The sensitivity of the twomultiplex assays with 40-cycle PCR was verified with onequinolone-sensitive strain of K. pneumoniae. The detection limit wasaround 10 copies of genomic DNA.

The complete assay for the specific detection of K. pneumoniae and itssusceptibility to quinolone contains the Klebsiella-specific primers(SEQ ID NOs. 1331 and 1332) described in Example 29 and either themultiplex containing the K. pneumoniae gyrA- and parC-specific primers(SEQ ID NOs. 1935, 1936, 1937) or the multiplex containing the K.pneumoniae gyrA- and parC-specific primers (SEQ ID NOs. 1934, 1937,1939, 1942). Amplification is coupled with post-PCR hybridization withthe internal probe specific to K. pneumoniae (SEQ ID NO. 2167) describedin Example 29 and the internal probes specific to wild-type K.pneumoniae gyrA and parC (SEQ ID NOs. 1943, 1944) and to the K.pneumoniae gyrA and parC variants (SEQ ID NOs. 1945-1949 and 1950-1953).

An assay was also developed for the detection of quinolone-resistant K.pneumoniae using the SmartCycler (Cepheid). Real-time detection is basedon the use of resistant K. pneumoniae gyrA-specific primers (SEQ ID NOs.1936 and 1937) and the K. pneumoniae-specific primers (SEQ ID NOs. 1331and 1332) described in Example 29. Internal probes were designed formolecular beacon detection of the wild-type K. pneumoniae gyrA (SEQ IDNO. 2251), for detection of the Ser-83 to Tyr or Phe and/or Asp-87 toGly or Asn in the GyrA subunit of DNA gyrase encoded by gyrA (SEQ IDNOs. 2250) and for detection of K. pneumoniae (SEQ ID NO. 2281).

Example 35

Development of a PCR Assay for Detection and Identification of S.pneumoniae and its Quinolone Resistance Genes gyrA and parC.

The analysis of gyrA and parC sequences from a variety of bacterialspecies revealed conserved regions allowing the design of PCR primersable to amplify the quinolone-resistance-determining region (QRDR) ofgyrA and parC from all S. pneumoniae strains. PCR primer pair SEQ IDNOs. 2040 and 2041 was designed to amplify the QRDR of S. pneumoniaegyrA, whereas PCR primer pair SEQ ID NOs. 2044 and 2045 was designed toamplify the QRDR of S. pneumoniae parC. The comparison of gyrA and parCsequences from S. pneumoniae strains with various levels of quinoloneresistance allowed the identification of amino acid substitutions Ser-81to Phe or Tyr in the GyrA subunit of DNA gyrase encoded by gyrA andamino acid changes Ser-79 to Phe in the ParC subunit of topoisomerase IVencoded by parC. These amino acid substitutions in the GyrA and ParCsubunits occur in isolates with intermediate- or high-level quinoloneresistance. Internal probes for the specific detection of each of thegyrA (SEQ ID NOs. 2042 and 2043) and parC (SEQ ID NO. 2046) mutationsidentified in quinolone-resistant S. pneumoniae were designed.

For all bacterial species, amplification was performed from purifiedgenomic DNA. 1 μl of genomic DNA at 0.1 ng/μL was transferred directlyto a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mMTris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.4 μM (each) of theabove primers SEQ ID NOs. 2040, 2041, 2044 and 2045, 0.05 mM bovineserum albumin (BSA) and 0.5 U Taq polymerase coupled with TaqStart™antibody. The optimal cycling conditions for maximum sensitivity andspecificity were 3 minutes at 95° C. for initial denaturation, thenforty cycles of two steps consisting of 1 second at 95° C. and 30seconds at 58° C., followed by terminal extension at 72° C. for 2minutes. In order to generate Digoxigenin (DIG)-labeled amplicons forcapture probe hybridization, 0.1×PCR DIG labeling four deoxynucleosidetriphosphates mix (Boehringer Mannheim GmbH) was used for amplification.

The DIG-labeled amplicons were hybridized to the capture probes bound to96-well plates. The plates were incubated with anti-DIG-alkalinephosphatase and the chemiluminescence was measured by using aluminometer (MLX, Dynex Technologies Inc.) after incubation with CSPDand recorded as Relative Light Unit (RLU). The RLU ratio of testedsample with and without captures probes was then calculated. A ratio 2.0was defined as a positive hybridization signal. All reactions wereperformed in duplicate.

The specificity of the multiplex assay with 40-cycle PCR was verified byusing 0.1 ng of purified genomic DNA from a panel of bacteria listed inTable 13. Strong amplification of both gyrA and parC was detected onlyfor the S. pneumoniae strains tested. Weak amplification of both gyrAand parC genes was detected for Staphylococcus simulans. The detectionlimit tested with purified genomic DNA from 5 strains of S. pneumoniaewas 1 to 10 genome copies. In addition, 5 quinolone-resistant and 2quinolone-sensitive clinical isolates of S. pneumoniae were tested tofurther validate the developed multiplex PCR coupled with capture probehybridization assays. There was a perfect correlation between detectionof S. pneumoniae gyrA and parC mutations and the susceptibility toquinolone.

The complete assay for the specific detection of S. pneumoniae and itssusceptibility to quinolone contains the S. pneumoniae-specific primers(SEQ ID NOs. 1179 and 1181) described in Example 20 and the multiplexcontaining the S. pneumoniae gyrA-specific and parC-specific primerpairs (SEQ ID NOS. 2040 and 2041 and SEQ ID NOs. 2044 and 2045).Amplification is coupled with post-PCR hybridization with the internalprobe specific to S. pneumoniae (SEQ ID NO. 1180) described in Exampleand the internal probes specific to each of the S. pneumoniae gyrA andparC variants (SEQ ID NOs. 2042, 2043 and 2046).

Example 36

Detection of Extended-Spectrum TEM-Type β-Lactamases in Escherichiacoli.

The analysis of TEM sequences which confer resistance tothird-generation cephalosporins and to β-lactamase inhibitors allowedthe identification of amino acid substitutions Met-69 to Ile or Leu orVal, Ser-130 to Gly, Arg-164 to Ser or His, Gly-238 to Ser, Glu-240 toLys and Arg-244 to Ser or Cys or Thr or His or Leu. PCR primers SEQ IDNOs. 1907 and 1908 were designed to amplify TEM sequences. Internalprobes for the specific detection of wild-type TEM (SEQ ID NO. 2141) andfor each of the amino acid substitutions (SEQ ID NOs. 1909-1926)identified in TEM variants were designed to detect resistance tothird-generation cephalosporins and to β-lactamase inhibitors. Designand synthesis of primers and probes, and detection of the hybridizationwere performed as described in Example 7.

For all bacterial species, amplification was performed from purifiedgenomic DNA. One μl of genomic DNA at 0.1 ng/μl was transferred directlyto a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mMTris-HCl (pH 9.0); 0.1% Triton X-100, 2.5 mM MgCl₂, 0.4 μM of theTEM-specific primers SEQ ID NOs. 1907 and 1908, 200 μM (each) of thefour deoxynucleoside triphosphates, 0.05 mM bovine serum albumin (BSA)and 0.5 U Taq polymerase (Promega) coupled with TaqStart™ antibody. PCRamplification and agarose gel analysis of the amplified products wereperformed as described in Example 28. The optimal cycling conditions formaximum sensitivity and specificity were 3 minutes at 95° C. for initialdenaturation, then forty cycles of three steps consisting of 5 secondsat 95° C., 30 seconds at 55° C. and 30 seconds at 72° C., followed byterminal extension at 72° C. for 2 minutes.

The specificity of the TEM-specific primers with 40-cycle PCR wasverified by using 0.1 ng of purified genomic from the followingbacteria: three third-generation cephalosporin-resistant Escherichiacoli strains (one with TEM-10, one with TEM-28 and the other withTEM-49), two third-generation cephalosporin-sensitive Escherichia colistrain (one with TEM-1 and the other without TEM), one third-generationcephalosporin-resistant Klebsiella pneumoniae strain (with TEM-47), andone β-lactamase-inhibitor-resistant Proteus mirabilis strain (withTEM-39). Amplification with the TEM-specific primers was detected onlyfor strains containing TEM.

The sensitivity of the assay with 40-cycle PCR was verified with threeE. coli strains containing TEM-1 or TEM-10 or TEM-49, one K. pneumoniaestrain containing TEM-47 and one P. mirabilis strain containing TEM-39.The detection limit was 5 to 100 copies of genomic DNA, depending on theTEM-containing strains tested.

The TEM-specific primers SEQ ID NOs. 1907 and 1908 were used inmultiplex with the Escherichia coli/Shigella sp.-specific primers SEQ IDNOs. 1661 and 1665 described in Example 28 to allow the completeidentification of Escherichia coli/Shigella sp. and the susceptibilityto β-lactams. PCR amplification with 0.4 μM of each of the primers andagarose gel analysis of the amplified products was performed asdescribed above.

The specificity of the multiplex with 40-cycle PCR was verified by using0.1 ng of purified genomic DNA from the following bacteria: threethird-generation cephalosporin-resistant Escherichia coli strains (onewith TEM-10, one with TEM-28 and the other with TEM-49), twothird-generation cephalosporin-sensitive Escherichia coli strain (onewith TEM-1 and the other without TEM), one third-generationcephalosporin-resistant Klebsiella pneumoniae strain (with TEM-47), andone β-lactamase-inhibitor-resistant Proteus mirabilis strain (withTEM-39). The multiplex was highly specific to Escherichia coli strainscontaining TEM.

The complete assay for detection of TEM-type β-lactamases in E. coliincludes PCR amplification using the multiplex containing theTEM-specific primers (SEQ ID NOs. 1907 and 1908) and the Escherichiacoli/Shigella sp.-specific primers (SEQ ID NOs. 1661 and 1665) coupledwith post PCR-hybridization with the internal probes specific towild-type TEM (SEQ ID NO. 2141) and to the TEM variants (SEQ ID NOs.1909-1926).

Example 37

Detection of Extended-Spectrum SHV-Type β-Lactamases in Klebsiellapneumoniae.

The comparison of SHV sequences, which confer resistance tothird-generation cephalosporins and to β-lactamase inhibitors, allowedthe identification of amino acid substitutions Ser-130 to Gly, Asp-179to Ala or Asn, Gly-238 to Ser, and Glu-240 to Lys. PCR primer pair SEQID NOs. 1884 and 1885 was designed to amplify SHV sequences. Internalprobes for the specific identification of wild-type SHV (SEQ ID NO.1896) and for each of the amino acid substitutions (SEQ ID NOs.1886-1895 and 1897-1898) identified in SHV variants were designed todetect resistance to third-generation cephalosporins and to β-lactamaseinhibitors. Design and synthesis of primers and probes, and detection ofthe hybridization were performed as described in Example 7.

For all bacterial species, amplification was performed from purifiedgenomic DNA. One μl of of genomic DNA at 0.1 ng/μl was transferreddirectly to a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl,10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.4 μM of theSHV-specific primers SEQ ID NO. 1884 and 1885, 200 μM (each) of the fourdeoxynucleoside triphosphates, 0.05 mM bovine serum albumin (BSA) and0.5 U Taq polymerase (Promega) coupled with TaqStart™ antibody. PCRamplification and agarose gel analysis of the amplified products wereperformed as described in Example 28. The optimal cycling conditions formaximum sensitivity and specificity were 3 minutes at 95° C. for initialdenaturation, then forty cycles of three steps consisting of 5 secondsat 95° C., 30 seconds at 55° C. and 30 seconds at 72° C., followed byterminal extension at 72° C. for 2 minutes.

The specificity of the SHV-specific primers with 40-cycle PCR wasverified by using 0.1 ng of purified genomic from the followingbacteria: two third-generation cephalosporin-resistant Klebsiellapneumoniae strains (one with SHV-2a and the other with SHV-12), onethird-generation cephalosporin-sensitive Klebsiella pneumoniae strain(with SHV-1), two third-generation cephalosporin-resistant Escherichiacoli strains (one with SHV-8 and the other with SHV-7), and twothird-generation cephalosporin-sensitive Escherichia coli strains (onewith SHV-1 and the other without any SHV). Amplification with theSHV-specific primers was detected only for strains containing SHV.

The sensitivity of the assay with 40-cycle PCR was verified with fourstrains containing SHV. The detection limit was 10 to 100 copies ofgenomic DNA, depending on the SHV-containing strains tested.

The amplification was coupled with post-PCR hybridization with theinternal probes specific for identification of wild-type SHV (SEQ ID NO.1896) and for each of the amino acid substitutions (SEQ ID NOs.1886-1895 and 1897-1898) identified in SHV variants. The specificity ofthe probes was verified with six strains containing various SHV enzymes,one Klebsiella pneumoniae strain containing SHV-1, one Klebsiellapneumoniae strain containing SHV-2a, one Klebsiella pneumoniae straincontaining SHV-12, one Escherichia coli strain containing SHV-1, oneEscherichia coli strain containing SHV-7 and one Escherichia coli straincontaining SHV-8. The probes correctly detected each of the SHV genesand their specific mutations. There was a perfect correlation betweenthe SHV genotype of the strains and the susceptibility to β-lactamantibiotics.

The SHV-specific primers SEQ ID NOs. 1884 and 1885 were used inmultiplex with the K. pneumoniae-specific primers SEQ ID NOs. 1331 and1332 described in Example 29 to allow the complete identification of K.pneumoniae and the susceptibility to β-lactams. PCR amplification with0.4 μM of each of the primers and agarose gel analysis of the amplifiedproducts were performed as described above.

The specificity of the multiplex with 40-cycle PCR was verified by using0.1 ng of purified genomic DNA from the following bacteria: three K.pneumoniae strains containing SHV-1, one Klebsiella pneumoniae straincontaining SHV-2a, one Klebsiella pneumoniae strain containing SHV-12,one K. rhinoscleromatis strain containing SHV-1, one Escherichia colistrain without SHV. The multiplex was highly specific to Klebsiellapneumoniae strain containing SHV.

Example 38

Development of a PCR Assay for the Detection and Identification ofNeisseria Gonorrhoeae and its Associated Tetracycline Resistance GenetetM.

The analysis of publicly available tetM sequences revealed conservedregions allowing the design of PCR primers specific to tetM sequences.The PCR primer pair SEQ ID NOs. 1588 and 1589 was used in multiplex withthe Neisseria gonorrhoeae-specific primers SEQ ID NOs. 551 and 552described in Example 31. Sequence alignment analysis of tetM sequencesrevealed regions suitable for the design of an internal probe specificto tetM (SEQ ID NO. 2254). PCR amplification was carried out on PTC-200thermocyclers (MJ Research) using 0.4 μM of each primer pair asdescribed in Example 28. The optimal cycling conditions for maximumsensitivity and specificity were as follow: three minutes at 95° C. forinitial denaturation, then forty cycles of two steps consisting of 1second at 95° C. and 30 seconds at 60° C., followed by terminalextension at 72° C. for 2 minutes.

The specificity of the multiplex PCR assay with 40-cycle PCR wasverified by using 0.1 ng of purified genomic DNA from the followingbacteria: two tetracycline-resistant Escherichia coli strains (onecontaining the tetracycline-resistant gene tetB and the other containingthe tetracycline-resistant gene tetC), one tetracycline-resistantPseudomonas aeruginosa strain (containing the tetracycline-resistantgene tetA), nine tetracycline-resistant Neisseria gonorrhoeae strains,two tetracycline-sensitive Neisseria meningitidis strains, onetetracycline-sensitive Neisseria polysaccharea strain, onetetracycline-sensitive Neisseria sicca strain and onetetracycline-sensitive Neisseria subflava strain. Amplification withboth the tetM-specific and Neisseria gonorrhoeae-specific primers wasdetected only for N. gonorrhoeae strains containing tetM. There was aweak amplification signal using Neisseria gonorrhoeae-specific primersfor the following species: Neisseria sicca, Neisseria polysaccharea andNeisseria meningitidis. There was a perfect correlation between the tetMgenotype and the tetracycline susceptibility pattern of the Neisseriagonorrhoeae strains tested. The internal probe specific to N.gonorrhoeae SEQ ID NO. 2166 described in Example 31 can discriminateNeisseria gonorrhoeae from the other Neisseria sp.

The sensitivity of the assay with 40-cycle PCR was verified with twotetracycline resistant strains of N. gonorrhoeae. The detection limitwas 5 copies of genomic DNA for both strains.

Example 39

Development of a PCR Assay for the Detection and Identification ofShigella sp. and their Associated Trimethoprim Resistance Gene DhfrIa.

The analysis of publicly available dhfrIa and other dhfr sequencesrevealed regions allowing the design of PCR primers specific to dhfrIasequences. The PCR primer pair (SEQ ID NOs. 1459 and 1460) was used inmultiplex with the Escherichia coli/Shigella sp.-specific primers SEQ IDNOs. 1661 and 1665 described in Example 28. Sequence alignment analysisof dhfrIa sequences revealed regions suitable for the design of aninternal probe specific to dhfrIa (SEQ ID NO. 2253). PCR amplificationand agarose gel analysis of the amplified products were performed asdescribed in Example 28 with an annealing temperature of 60° C. Thespecificity of the multiplex assay with 40-cycle PCR was verified byusing 0.1 ng of purified genomic DNA from a panel of bacteria. The listincluded the following trimethoprim-sensitive strains, Salmonellatyphimyurium, Salmonella typhi, Salmonella enteritidis, Tatumellaptyseos, Klebsiella pneumoniae, Enterobacter aerogenes, Citrobacterfarmeri, Campylobacter jejuni, Serratia marcescens, Shigelladysenteriae, Shigella flexneri, Shigella sonnei, sixtrimethoprim-resistant Escherichia coli strains (containing dhfrIa ordhfrV or dhfrVII or dhfrXII or dhfrXIII or dhfrXV), fourtrimethoprim-resistant strains containing dhfrIa (Shigella sonnei,Shigella flexneri, Shigella dysenteriae and Escherichia coli). There wasa perfect correlation between the dhfrIa genotype and the trimethoprimsusceptibility pattern of the Escherichia coli and Shigella sp. strainstested. The dhfrIa primers were specific to the dhfrIa gene and did notamplify any of the other trimethoprim-resistant dhfr genes tested. Thesensitivity of the multiplex assay with 40-cycle PCR was verified withthree strains of trimethoprim-resistant strains of Shigella sp. Thedetection limit was 5 to 10 genome copies of DNA, depending on theShigella sp. strains tested.

Example 40

Development of a PCR Assay for the Detection and Identification ofAcinetobacter Baumannii and its Associated Aminoglycoside ResistanceGene aph(3′)-VIa.

The comparison of publicly available aph(3)-VIa sequence revealedregions allowing the design of PCR primers specific to aph(3)-VIa. ThePCR primer pair (SEQ ID NOs. 1404 and 1405) was used in multiplex withthe Acinetobacter baumannii-specific primers SEQ ID NOs. 1692 and 1693described in Example 30. Analysis of the aph(3)-VIa sequence revealedregion suitable for the design of an internal probe specific toaph(3)-VIa (SEQ ID NO. 2252). PCR amplification and agarose gel analysisof the amplified products were performed as described in Example 28. Thespecificity of the multiplex assay with 40-cycle PCR was verified byusing 0.1 ng of purified genomic DNA from a panel of bacteria including:two aminoglycoside-resistant A. baumanni strains (containingaph(3)-VIa), one aminoglycoside-sensitive A. baumani strain, one of eachof the following aminoglycoside-resistant bacteria, one Serratiamarcescens strain containing the aminoglycoside-resistant gene aacC1,one Serratia marcescens strain containing the aminoglycoside-resistantgene aacC4, one Enterobacter cloacae strain containing theaminoglycoside-resistant gene aacC2, one Enterococcus faecaliscontaining the aminoglycoside-resistant gene aacA-aphD, one Pseudomonasaeruginosa strain containing the aminoglycoside-resistant gene aac6IIaand one of each of the following aminoglycoside-sensitive bacterialspecies, Acinetobacter anitratus, Acinetobacter lwoffi, Psychobbacterphenylpyruvian, Neisseria gonorrhoeae, Haemophilus haemolyticus,Haemophilus influenzae, Yersinia enterolitica, Proteus vulgaris,Eikenella corrodens, Escherichia coli. There was a perfect correlationbetween the aph(3)-VIa genotype and the aminoglycoside susceptibilitypattern of the A. baumannii strains tested. The aph(3)-VIa-specificprimers were specific to the aph(3)-VIa gene and did not amplify any ofthe other aminoglycoside-resistant genes tested. The sensitivity of themultiplex assay with 40-cycle PCR was verified with two strains ofaminoglycoside-resistant strains of A. baumannii. The detection limitwas 5 genome copies of DNA for both A. baumannii strains tested.

Example 41

Specific Identification of Bacteroides fragilis Using atpD (V-Type)Sequences.

The comparison of atpD (V-type) sequences from a variety of bacterialspecies allowed the selection of PCR primers for Bacteroides fragilis.The strategy used to design the PCR primers was based on the analysis ofa multiple sequence alignment of various atpD sequences from B.fragilis, as well as atpD sequences from the related species B. dispar,bacterial genera and archaea, especially representatives withphylogenetically related atpD sequences. A careful analysis of thisalignment allowed the selection of oligonucleotide sequences which areconserved within the target species but which discriminate sequencesfrom other species, especially from closely related species B. dispar,thereby permitting the species-specific and ubiquitous detection andidentification of the target bacterial species.

The chosen primer pair, SEQ ID NOs. 2134-2135, produces an amplificationproduct of 231 bp. Standard PCR was carried out on PTC-200 thermocyclers(MJ Research Inc.) using 0.4 μM of each primers pair as described inExample 28. The optimal cycling conditions for maximum sensitivity andspecificity were as follows: three minutes at 95° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 60° C., followed by terminal extension at 72°C. for 2 minutes.

The format of this assay is not limited to the one described above. Aperson skilled in the art could adapt the assay for different formatssuch as PCR with real-time detection using molecular beacon probes.Molecular beacon probes designed to be used in this assay include, butare not limited to, SEQ ID NO. 2136 for the detection of the B. fragilisamplicon.

Example 42

Evidence for Horizontal Gene Transfer in the Evolution of the ElongationFactor Tu in Enterococci.

Overview

The elongation factor Tu, encoded by tuf genes, is a GTP binding proteinthat plays a central role in protein synthesis. One to three tuf genesper genome are present depending on the bacterial species. Most low G+Cgram-positive bacteria carry only one tuf gene. We have designeddegenerate PCR primers derived from consensus sequences of the tuf geneto amplify partial tuf sequences from 17 enterococcal species and otherphylogenetically related species. The amplified DNA fragments weresequenced either by direct sequencing or by sequencing cloned insertscontaining putative amplicons. Two different tuf genes (tufA and tufB)were found in 11 enterococcal species, including Enterococcus avium, E.casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E.hirae, E. malodoratus, E. mundtii, E. pseudoavium, and E. raffinosus.For the other six enterococcal species (E. cecorum, E. columbae, E.faecalis, E. sulfureus, E. saccharolyticus, and E. solitarius), only thetufA gene was present. Based on 16S rRNA gene sequence analysis, the 11species having two tuf genes all share a common ancestor, while the sixspecies having only one copy diverged from the enterococcal lineagebefore that common ancestor. The presence of one or two copies of thetuf gene in enterococci was confirmed by Southern hybridization.Phylogenetic analysis of tuf sequences demonstrated that theenterococcal tufA gene branches with the Bacillus, Listeria andStaphylococcus genera, while the enterococcal tufB gene clusters withthe genera Streptococcus and Lactococcus. Primary structure analysisshowed that four amino acid residues within the sequenced regions areconserved and unique to the enterococcal tufB genes and the tuf genes ofstreptococci and L. lactis. The data suggest that an ancestralstreptococcus or a streptococcus-related species may have horizontallytransferred a tuf gene to the common ancestor of the 11 enterococcalspecies which now carry two tuf genes.

Introduction

The elongation factor Tu (EF-Tu) is a GTP binding protein playing acentral role in protein synthesis. It mediates the recognition andtransport of aminoacyl-tRNAs and their positioning to the A-site of theribosome. The highly conserved function and ubiquitous distributionrender the elongation factor a valuable phylogenetic marker amongeubacteria and even throughout the archaebacterial and eukaryotickingdoms. The tuf genes encoding elongation factor Tu are present invarious copy numbers per bacterial genome. Most gram-negative bacteriacontain two tuf genes. As found in Escherichia coli, the two genes,while being almost identical in sequence, are located in different partsof the bacterial chromosome. However, recently completed microbialgenomes revealed that only one tuf gene is found in Helicobacter pylorias well as in some obligate parasitic bacteria, such as Borreliaburgdorferi, Rickettsia prowazekii, and Treponema pallidum, and in somecyanobacteria. In most gram-positive bacteria studied so far, only onetuf gene was found. However, Southern hybridization showed that thereare two tuf genes in some clostridia as well as in Streptomycescoelicolor and S. lividans. Up to three tuf-like genes have beenidentified in S. ramocissimus.

Although massive prokaryotic gene transfer is suggested to be one of thefactors responsible for the evolution of bacterial genomes, the genesencoding components of the translation machinery are thought to behighly conserved and difficult to be transferred horizontally due to thecomplexity of their interactions. However, a few recent studiesdemonstrated evidence that horizontal gene transfer has also occurred inthe evolution of some genes coding for the translation apparatus,namely, 16S rRNA and some aminoacyl-tRNA synthetases. No further datasuggest that such a mechanism is involved in the evolution of theelongation factors. Previous studies concluded that the two copies oftuf genes in the genomes of some bacteria resulted from an ancient eventof gene duplication. Moreover, a study of the tuf gene in R. prowazekiisuggested that intrachromosomal recombination has taken place in theevolution of the genome of this organism.

To date, little is known about the tuf genes of enterococcal species. Inthis study, we analyzed partial sequences of tuf genes in 17enterococcal species, namely, E. avium, E. casseliflavus, E. cecorum, E.columbae, E. dispar, E. durans, E. faecalis, E. faecium, E. gallinarum,E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, E. raffinosus, E.saccharolyticus, E. solitarius, and E. sulfureus. We report here thepresence of two divergent copies of tuf genes in 11 of theseenterococcal species. The 6 other species carried a single tuf gene. Theevolutionary implications are discussed.

Materials and Methods

Bacterial Strains.

Seventeen enterococcal strains and other gram-positive bacterial strainsobtained from the American Type Culture Collection (ATCC, Manassas, Va.)were used in this study (Table 16). All strains were grown on sheepblood agar or in brain-heart infusion broth prior to DNA isolation.

DNA Isolation.

Bacterial DNAs were prepared using the G NOME DNA extraction kit(Bio101, Vista, Calif.) as previously described.

Sequencing of Putative tuf Genes.

In order to obtain the tuf gene sequences of enterococci and othergram-positive bacteria, two sequencing approaches were used: 1)sequencing of cloned PCR products and 2) direct sequencing of PCRproducts. A pair of degenerate primers (SEQ ID NOs. 664 and 697) wereused to amplify an 886-bp portion of the tuf genes from enterococcalspecies and other gram-positive bacteria as previously described. For E.avium, E. casseliflavus, E. dispar, E. durans, E. faecium, E.gallinarum, E. hirae, E. mundtii, E. pseudoavium, and E. raffinosus, theamplicons were cloned using the Original TA cloning kit (Invitrogen,Carlsbad, Calif.) as previously described. Five clones for each specieswere selected for sequencing. For E. cecorum, E. faecalis, E.saccharolyticus, and E. solitarius as well as the other gram-positivebacteria, the sequences of the 886-bp amplicons were obtained by directsequencing. Based on the results obtained from the earlier rounds ofsequencing, two pairs of primers were designed for obtaining the partialtuf sequences from the other enterococcal species by direct sequencing.One pair of primers (SEQ ID NOs. 543 and 660) were used to amplify theenterococcal tuf gene fragments from E. columbae, E. malodoratus, and E.sulfureus. Another pair of primers (SEQ ID NOs. 664 and 661) were usedto amplify the second tuf gene fragments from E. avium, E. malodoratus,and E. pseudoavium.

Prior to direct sequencing, PCR products were electrophoresed on 1%agarose gel at 120V for 2 hours. The gel was then stained with 0.02%methylene blue for 30 minutes and washed twice with autoclaved distilledwater for 15 minutes. The gel slices containing PCR products of theexpected sizes were cut out and purified with the QIAquick gelextraction kit (QIAgen Inc., Mississauga, Ontario, Canada) according tothe manufacturer's instructions. PCR mixtures for sequencing wereprepared as described previously. DNA sequencing was carried out withthe Big Dye Terminator Ready Reaction cycle sequencing kit using a 377DNA sequencer (PE Applied Biosystems, Foster City, Calif.). Both strandsof the amplified DNA were sequenced. The sequence data were verifiedusing the Sequencer™ 3.0 software (Gene Codes Corp., Ann Arbor, Mich.).

Sequence Analysis and Phylogenetic Study.

Nucleotide sequences of the tuf genes and their respective flankingregions for E. faecalis, Staphylococcus aureus, and Streptococcuspneumoniae, were retrieved from the TIGR microbial genome database andS. pyogenes from the University of Oklahoma database. DNA sequences anddeduced protein sequences obtained in this study were compared withthose in all publicly available databases using the BLAST and FASTAprograms. Unless specified, sequence analysis was conducted with theprograms from GCG package (Version 10; Genetics Computer Group, Madison,Wis.). Sequence alignment of the tuf genes from 74 species representingall three kingdoms of life (Tables 16 and 17) were carried out by use ofPileup and corrected upon visual analysis. The N- and C-terminiextremities of the sequences were trimmed to yield a common block of 201amino acids sequences and equivocal residues were removed. Phylogeneticanalysis was performed with the aid of PAUP 4.0b4 written by Dr. DavidL. Swofford (Sinauer Associates, Inc., Publishers, Sunderland, Mass.).The distance matrix and maximum parsimony were used to generatephylogenetic trees and bootstrap resampling procedures were performedusing 500 and 100 replications in each analysis, respectively.

Protein Structure Analysis.

The crystal structures of (i) Thermus aquaticus EF-Tu in complex withPhe-tRNA^(Phe) and a GTP analog and (ii) E. coli EF-Tu in complex withGDP served as templates for constructing the equivalent models forenterococcal EF-Tu. Homology modeling of protein structure was performedusing the SWISS-MODEL server and inspected using the SWISS-PDB viewerversion 3.1.

Southern Hybridization.

In a previous study, we amplified and cloned an 803-bp PCR product ofthe tuf gene fragment from E. faecium. Two divergent sequences of theinserts, which we assumed to be tufA and tufB genes, were obtained. Therecombinant plasmid carrying either tufA or tufB sequence was used togenerate two probes labeled with Digoxigenin (DIG)-11-dUTP by PCRincorporation following the instructions of the manufacturer (BoehringerMannheim, Laval, Québec, Canada). Enterococcal genomic DNA samples (1-2μg) were digested to completion with restriction endonucleases BglII andXbaI as recommended by the supplier (Amersham Pharmacia Biotech,Mississauga, Ontario, Canada). These restriction enzymes were chosenbecause no restriction sites were observed within the amplified tuf genefragments of most enterococci. Southern blotting and filterhybridization were performed using positively charged nylon membranes(Boehringer Mannheim) and QuikHyb hybridization solution (StratageneCloning Systems, La Jolla, Calif.) according to the manufacturers'instructions with modifications. Twenty μ1 of each digestion wereelectrophoresed for 2 h at 120V on a 0.8% agarose gel. The DNA fragmentswere denatured with 0.5 M NaOH and transferred by Southern blotting ontoa positively charged nylon membrane (Boehringer Mannheim). The filterswere pre-hybridized for 15 min and then hybridized for 2 h in theQuikHyb solution at 68° C. with either DIG-labeled probe.Posthybridization washings were performed twice with 0.5×SSC, 1% SDS atroom temperature for 15 min and twice in the same solution at 60° C. for15 min. Detection of bound probes was achieved using disodium3-(4-methoxyspiro (1,2-dioxetane-3,2′-(5′-chloro)tricyclo(3,3.1.1^(3.7)) decan)-4-yl) phenyl phosphate (CSPD) (BoehringerMannheim) as specified by the manufacturer.

GenBank Submission.

The GenBank accession numbers for partial tuf gene sequences generatedin this study are given in Table 16.

Results

Sequencing and Nucleotide Sequence Analysis.

In this study, all gram-positive bacteria other than enterococci yieldeda single tuf sequence of 886 bp using primers SEQ ID NOs. 664 and 697(Table 16). Each of four enterococcal species including E. cecorum, E.faecalis, E. saccharolyticus, and E. solitarius also yielded one 886-bptuf sequence. On the other hand, for E. avium, E. casseliflavus, E.dispar, E. durans, E. faecium, E. gallinarum, E. hirae, E. mundtii, E.pseudoavium, and E. raffinosus, direct sequencing of the 886-bpfragments revealed overlapping peaks according to their sequencechromatograms, suggesting the presence of additional copies of the tufgene. Therefore, the tuf gene fragments of these 10 species were clonedfirst and then sequenced. Sequencing data revealed that two differenttypes of tuf sequences (tufA and tufB) are found in eight of thesespecies including E. casseliflavus, E. dispar, E. durans, E. faecium, E.gallinarum, E. hirae, E. mundtii, and E. raffinosus. Five clones from E.avium and E. pseudoavium yielded only a single tuf sequence. These newsequence data allowed the design of new primers specific for theenterococcal tufA or tufB sequences. Primers SEQ ID NOs. 543 and 660were designed to amplify only enterococcal tufA sequences and a 694-bpfragment was amplified from all 17 enterococcal species. The 694-bpsequences of tufA genes from E. columbae, E. malodoratus, and E.sulfureus were obtained by direct sequencing using these primers.Primers SEQ ID NOs. 664 and 661 were designed for the amplification of730-bp portion of tufB genes and yielded the expected fragments from 11enterococcal species, including E. malodoratus and the 10 enterococcalspecies in which heterogeneous tuf sequences were initially found. Thesequences of the tufB fragments for E. avium, E. malodoratus and E.pseudoavium were determined by direct sequencing using the primers SEQID NOs. 664 and 661. Overall, tufA gene fragments were obtained from all17 enterococcal species but tufB gene fragments were obtained with only11 enterococcal species (Table 16).

The identities between tufA and tufB for each enterococcal species were68-79% at the nucleotide level and 81 to 89% at the amino acid level.The tufA gene is highly conserved among all enterococcal species withidentities varying from 87% to 99% for DNA and 93% to 99% for amino acidsequences, while the identities among tufB genes of enterococci variesfrom 77% to 92% for DNA and 91% to 99% for amino acid sequences,indicating their different origins and evolution (FIG. 26). Since E.solitarius has been transferred to the genus Tetragenococcus, which isalso a low G+C gram-positive bacterium, our sequence comparison did notinclude this species as an enterococcus. G+C content of enterococcaltufA sequences ranged from 40.8% to 43.1%, while that of enterococcaltufB sequences varied from 37.8% to 46.3%. Based on amino acid sequencecomparison, the enterococcal tufA gene products share higher identitieswith those of Abiotrophia adiacens, Bacillus subtilis, Listeriamonocytogenes, S. aureus, and S. epidermidis. On the other hand, theenterococcal tufB gene products share higher percentages of amino acididentity with the tuf genes of S. pneumoniae, S. pyogenes andLactococcus lactis (FIG. 26).

In order to elucidate whether the two enterococcal tuf sequences encodegenuine EF-Tu, the deduced amino acid sequences of both genes werealigned with other EF-Tu sequences available in SWISSPROT (Release 38).Sequence alignment demonstrated that both gene products are highlyconserved and carry all conserved residues present in this portion ofprokaryotic EF-Tu (FIG. 4). Therefore, it appears that both geneproducts could fulfill the function of EF-Tu. The partial tuf genesequences encode the portion of EF-Tu from residues 117 to 317, numberedas in E. coli. This portion makes up of the last four α-helices and twoβ-strands of domain I, the entire domain II and the N-terminal part ofdomain III on the basis of the determined structures of E. coli EF-Tu.

Based on the deduced amino acid sequences, the enterococcal tufB geneshave unique conserved residues Lys129, Leu140, Ser230, and Asp234 (E.coli numbering) that are also conserved in streptococci and L. lactis,but not in the other bacteria (FIG. 4). All these residues are locatedin loops except for Ser230. In other bacteria the residue Ser230 issubstituted for highly conserved Thr, which is the 5^(th) residue of thethird β-strand of domain II. This region is partially responsible forthe interaction between the EF-Tu and aminoacyl-tRNA by the formation ofa deep pocket for any of the 20 naturally occurring amino acids.According to our three-dimensional model (data not illustrated), thesubstitution Thr230→Ser in domain II of EF-Tu may have little impact onthe capability of the pocket to accommodate any amino acid. However, thehigh conservation of Thr230 comparing to the unique Ser substitutionfound only in streptococci and 11 enterococci could suggest a subtlefunctional role for this residue.

The tuf gene sequences obtained for E. faecalis, S. aureus, S.pneumoniae and S. pyogenes were compared with their respectiveincomplete genome sequence. Contigs with more than 99% identity wereidentified. Analysis of the E. faecalis genome data revealed that thesingle E. faecalis tuf gene is located within an str operon where tuf ispreceded by fus that encodes the elongation factor G. This str operon ispresent in S. aureus and B. subtilis but not in the two streptococcalgenomes examined. The 700-bp or so sequence upstream the S. pneumoniaetuf gene has no homology with any known gene sequences. In S. pyogenes,the gene upstream of tuf is similar to a cell division gene, ftsW,suggesting that the tuf genes in streptococci are not arranged in a stroperon.

Phylogenetic Analysis.

Phylogenetic analysis of the tuf amino acid sequences withrepresentatives of eubacteria, archeabacteria, and eukaryotes usingneighbor-joining and maximum parsimony methods showed three majorclusters representing the three kingdoms of life. Both methods gavesimilar topologies consistent with the rRNA gene data (data not shown).Within the bacterial clade, the tree is polyphyletic but tufA genes fromall enterococcal species always clustered with those from other low G+Cgram-positive bacteria (except for streptococci and lactococci), whilethe tufB genes of the 11 enterococcal species form a distinct clusterwith streptococci and L. lactis (FIG. 5). Duplicated genes from the sameorganism do not cluster together, thereby not suggesting evolution byrecent gene duplication.

Southern Hybridization.

Southern hybridization of BglII/XbaI digested genomic DNA from 12enterococcal species tested with the tufA probe (DIG-labeled tufAfragment from E. faecium) yielded two bands of different sizes in 9species, which also carried two divergent tuf sequences according totheir sequencing data. For E. faecalis and E. solitarius, a single bandwas observed indicating that one tuf gene is present (FIG. 6). A singleband was also found when digested genomic DNA from S. aureus, S.pneumoniae, and S. pyogenes were hybridized with the tufA probe (datanot shown). For E. faecium, the presence of three bands can be explainedby the existence of a XbaI restriction site in the middle of the tufAsequence, which was confirmed by sequencing data. Hybridization with thetufB probe (DIG-labeled tufB fragment of E. faecium) showed a bandingprofile similar to the one obtained with the tufA probe (data notshown).

Discussion

In this study, we have shown that two divergent copies of genes encodingthe elongation factor Tu are present in some enterococcal species.Sequence data revealed that both genes are highly conserved at the aminoacid level. One copy (tufA) is present in all enterococcal species,while the other (tufB) is present only in 11 of the 17 enterococcalspecies studied. Based on 16S rRNA sequence analysis, these 11 speciesare members of three different enterococcal subgroups (E. avium, E.faecium, and E. gallinarum species groups) and a distinct species (E.dispar). Moreover, 16S rDNA phylogeny suggests that these 11 speciespossessing 2 tuf genes all share a common ancestor before they furtherevolved to become the modern species. Since the six other species havingonly one copy diverged from the enterococcal lineage before that commonancestor, it appears that the presence of one tuf gene in these sixspecies is not attributable to gene loss.

Two clusters of low G+C gram-positive bacteria were observed in thephylogenetic tree of the tuf genes: one contains a majority of low G+Cgram-positive bacteria and the other contains lactococci andstreptococci. This is similar to the finding on the basis ofphylogenetic analysis of the 16S rRNA gene and the hrcA gene coding fora unique heat-shock regulatory protein. The enterococcal tufA genesbranched with most of the low G+C gram-positive bacteria, suggestingthat they originated from a common ancestor. On the other hand, theenterococcal tufB genes branched with the genera Streptococcus andLactococcus that form a distinct lineage separated from other low G+Cgram-positive bacteria (FIG. 5). The finding that these EF-Tu proteinsshare some conserved amino acid residues unique to this branch alsosupports the idea that they may share a common ancestor. Although theseconserved residues might result from convergent evolution upon aspecialized function, such convergence at the sequence level, even for afew residues, seems to be rare, making it an unlikely event. Moreover,no currently known selective pressure, if any, would account for keepingone versus two tuf genes in bacteria. The G+C contents of enterococcaltufA and tufB sequences are similar, indicating that they bothoriginated from low G+C gram-positive bacteria, in accordance with thephylogenetic analysis.

The tuf genes are present in various copy numbers in different bacteria.Furthermore, the two tuf genes are normally associated withcharacteristic flanking genes. The two tuf gene copies commonlyencountered within gram-negative bacteria are part of the bacterial stroperon and tRNA-tufB operon, respectively. The arrangement of tufA inthe str operon was also found in a variety of bacteria, includingThermotoga maritima, the most ancient bacteria sequenced so far, Aquifexaeolicus, cyanobacteria, Bacillus sp., Micrococcus luteus, Mycobacteriumtuberculosis, and Streptomyces sp. Furthermore, the tRNA-tufB operon hasalso been identified in Aquifex aeolicus, Thermus thermophilus, andChlamydia trachomatis. The two widespread tuf gene arrangements argue infavor of their ancient origins. It is noteworthy that most obligateintracellular parasites, such as Mycoplasma sp., R. prowazekii, B.burgdorferi, and T. pallidum, contain only one tuf gene. Their flankingsequences are distinct from the two conserved patterns as a result ofselection for effective propagation by an extensive reduction in genomesize by intragenomic recombination and rearrangement.

Most gram-positive bacteria with low G+C content sequenced to datecontain only a single copy of the tuf gene as a part of the str operon.This is the case for B. subtilis, S. aureus and E. faecalis. PCRamplification using a primer targeting a conserved region of the fusgene and the tufA-specific primer SEQ ID NO. 660, but not thetufB-specific primer SEQ ID NO. 661, yielded the expected amplicons forall 17 enterococcal species tested, indicating the presence of thefus-tuf organization in all enterococci (data not shown). However, inthe genomes of S. pneumoniae and S. pyogenes, the sequences flanking thetuf genes varies although the tuf gene itself remains highly conserved.The enterococcal tufB genes are clustered with streptococci, but atpresent we do not have enough data to identify the genes flanking theenterococcal tufB genes. Furthermore, the functional role of theenterococcal tufB genes remains unknown. One can only postulate that thetwo divergent gene copies are expressed under different conditions.

The amino acid sequence identities between the enterococcal tufA andtufB genes are lower than either i) those between the enterococcal tufAand the tuf genes from other low G+C gram-positive bacteria(streptococci and lactococci excluded) or ii) those between theenterococcal tufB and streptococcal and lactococcal tuf genes. Thesefindings suggest that the enterococcal tufA genes share a commonancestor with other low G+C gram-positive bacteria via the simple schemeof vertical evolution, while the enterococcal tufB genes are moreclosely related to those of streptococci and lactococci. The facts thatsome enterococci possess an additional tuf gene and that the singlestreptococcal tuf gene is not clustered with other low G+C gram-positivebacteria cannot be explained by the mechanism of gene duplication orintrachromosomal recombination. According to sequence and phylogeneticanalysis, we propose that the presence of the additional copy of the tufgenes in 11 enterococcal species is due to horizontal gene transfer. Thecommon ancestor of the 11 enterococcal species now carrying tufB genesacquired a tuf gene from an ancestral streptococcus or astreptococcus-related species during enterococcal evolution through genetransfer before the diversification of modern enterococci. Further studyof the flanking regions of the gene may provide more clues for theorigin and function of this gene in enterococci.

Recent studies of genes and genomes have demonstrated that considerablehorizontal transfer occurred in the evolution of aminoacyl-tRNAsynthetases in all three kingdoms of life. The heterogeneity of 16S rRNAis also attributable to horizontal gene transfer in some bacteria, suchas Streptomyces, Thermomonospora chromogena and Mycobacterium celatum.In this study, we provide the first example in support of a likelyhorizontal transfer of the tuf gene encoding the elongation factor Tu.This may be an exception since stringent functional constraints do notallow for frequent horizontal transfer of the tuf gene as with othergenes. However, enterococcal tuf genes should not be the only suchexception as we have noticed that the phylogeny of Streptomyces tufgenes is equally or more complex than that of enterococci. For example,the three tuf-like genes in a high G+C gram-positive bacterium, S.ramocissimus, branched with the tuf genes of phylogenetically divergentgroups of bacteria (FIG. 5). Another example may be the tuf genes inclostridia, which represent a phylogenetically very broad range oforganisms and form a plethora of lines and groups of variouscomplexities and depths. Four species belonging to three differentclusters within the genus Clostridium have been shown by Southernhybridization to carry two copies of the tuf gene. Further sequence dataand phylogenetic analysis may help interpreting the evolution of theelongation factor Tu in these gram-positive bacteria. Since the tufgenes and 16S rRNA genes are often used for phylogenetic study, theexistence of duplicate genes originating from horizontal gene transfermay alter the phylogeny of microorganisms when the laterally acquiredcopy of the gene is used for such analysis. Hence, caution should betaken in interpreting phylogenetic data. In addition, the two tuf genesin enterococci have evolved separately and are distantly related to eachother phylogenetically. The enterococcal tufB genes are less conservedand unique to the 11 enterococcal species only. We previouslydemonstrated that the enterococcal tufA genes could serve as a target todevelop a DNA-based assay for identification of enterococci. Theenterococcal tufB genes would also be useful in identification of these11 enterococcal species.

Example 43

Elongation Factor Tu (Tuf) and the F-ATPase Beta-Subunit (atpD) asPhylogenetic Tools for Species of the Family Enterobacteriaceae.

Summary

The phylogeny of enterobacterial species commonly found in clinicalsamples was analyzed by comparing partial sequences of their elongationfactor Tu (tuf) genes and their F-ATPase beta-subunit (atpD) genes. A884-bp fragment for tuf and a 884- or 871-bp fragment for atpD weresequenced for 88 strains of 72 species from 25 enterobacterial genera.The atpD sequence analysis revealed a specific indel to Pantoea andTatumella species showing for the first time a tight phylogeneticaffiliation between these two genera. Comprehensive tuf and atpDphylogenetic trees were constructed and are in agreement with eachother. Monophyletic genera are Yersinia, Pantoea, Edwardsiella, Cedecea,Salmonella, Serratia, Proteus, and Providencia. Analogous trees wereobtained based on available 16S rDNA sequences from databases. tuf andatpD phylogenies are in agreement with the 16S rDNA analysis despite thesmaller resolution power for the latter. In fact, distance comparisonsrevealed that tuf and atpD genes provide a better resolution for pairsof species belonging to the family Enterobacteriaceae. However, 16S rDNAdistances are better resolved for pairs of species belonging todifferent families. In conclusion, tuf and atpD conserved genes aresufficiently divergent to discriminate different species inside thefamily Enterobacteriaceae and offer potential for the development ofdiagnostic tests based on DNA to identify enterobacterial species.

Introduction

Members of the family Enterobacteriaceae are facultatively anaerobicgram-negative rods, catalase-positive and oxydase-positive (Brenner,1984). They are found in soil, water, plants, and in animals frominsects to man. Many enterobacteria are opportunistic pathogens. Infact, members of this family are responsible for about 50% of nosocomialinfections in the United States (Brenner, 1984). Therefore, this familyis of considerable clinical importance.

Major classification studies on the family Enterobacteriaceae are basedon phenotypic traits (Brenner et al., 1999; Brenner et al., 1980; Dickey& Zumoff, 1988; Farmer III et al., 1980; Farmer III et al., 1985b;Farmer III et al., 1985a) such as biochemical reactions andphysiological characteristics. However, phenotypically distinct strainsmay be closely related by genotypic criteria and may belong to the samegenospecies (Bercovier et al., 1980; Hartl & Dykhuizen, 1984). Also,phenotypically close strains (biogroups) may belong to differentgenospecies, like Klebsiella pneumoniae and Enterobacter aerogenes(Brenner, 1984) for example. Consequently, identification andclassification of certain species may be ambiguous with techniques basedon phenotypic tests (Janda et al., 1999; Kitch et al., 1994; Sharma etal., 1990).

More advances in the classification of members of the familyEnterobacteriaceae have come from DNA-DNA hybridization studies (Brenneret al., 1993; Brenner et al., 1986; Brenner, et al., 1980; Farmer III,et al., 1980; Farmer III, et al., 1985b; Izard et al., 1981; Steigerwaltet al., 1976). Furthermore, the phylogenetic significance of bacterialclassification based on 16S rDNA sequences has been recognized by manyworkers (Stackebrandt & Goebel, 1994; Wayne et al., 1987). However,members of the family Enterobacteriaceae have not been subjected toextensive phylogenetic analysis of 16S rDNA (Sproer et al., 1999). Infact, this molecule was not thought to solve taxonomic problemsconcerning closely related species because of its very high degree ofconservation (Brenner, 1992; Sproer, et al., 1999). Another drawback ofthe 16S rDNA gene is that it is found in several copies within thegenome (seven in Escherichia coli and Salmonella typhimurium) (Hill &Harnish, 1981). Due to sequence divergence between the gene copies,direct sequencing of PCR products is often not suitable to achieve arepresentative sequence (Cilia et al., 1996; Hill & Harnish, 1981).Other genes such as gap and ompA (Lawrence et al., 1991), rpoB (Molletet al., 1997), and infB (Hedegaard et al., 1999) were used to resolvethe phylogeny of enterobacteria. However, none of these studies coveredan extensive number of species.

tuf and atpD are the genes encoding the elongation factor Tu (EF-Tu) andthe F-ATPase beta-subunit, respectively. EF-Tu is involved in peptidechain formation (Ludwig et al., 1990). The two copies of the tuf gene(tufA and tufB) found in enterobacteria (Sela et al., 1989) share highidentity level (99%) in Salmonella typhimurium and in E. coli. Therecombination phenomenon could explain sequence homogenization betweenthe two copies (Abdulkarim & Hughes, 1996; Grunberg-Manago, 1996).F-ATPase is present on the plasma membranes of eubacteria (Nelson &Taiz, 1989). It functions mainly in ATP synthesis (Nelson & Taiz, 1989)and the beta-subunit contains the catalytic site of the enzyme. EF-Tuand F-ATPase are highly conserved throughout evolution and showsfunctional constancy (Amann et al., 1988; Ludwig, et al., 1990).Recently, phylogenies based on protein sequences from EF-Tu and F-ATPasebeta-subunit showed good agreement with each other and with the rDNAdata (Ludwig et al., 1993).

We elected to sequence 884-bp fragments of tuf and atpD from 88clinically relevant enterobacterial strains representing 72 species from25 genera. These sequences were used to create phylogenetic trees thatwere compared with 16S rDNA trees. These trees revealed good agreementwith each others and demonstrated the high resolution of tuf and atpDphylogenies at the species level.

Materials and Methods

Bacterial Strains and Genomic Material.

All bacterial strains used in this study were obtained from the AmericanType Culture Collection (ATCC) or the Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSMZ). These enterobacteria canall be recovered from clinical specimens, but not all are pathogens.Whenever possible, we choose type strains. Identification of all strainswas confirmed by classical biochemical tests using the automated systemMicroScan WalkAway-96 system equipped with a Negative BP Combo PanelType 15 (Dade Behring Canada). Genomic DNA was purified using the G NOMEDNA kit (Bio 101). Genomic DNA from Yersinia pestis was kindly providedby Dr. Robert R. Brubaker. Strains used in this study and theirdescriptions are shown in Table 19.

PCR Primers.

The eubacterial tuf and atpD gene sequences available from publicdatabases were analyzed using the GCG package (version 8.0) (GeneticsComputer Group). Based on multiple sequence alignments, two highlyconserved regions were chosen for each genes, and PCR primers werederived from these regions with the help of Oligo primer analysissoftware (version 5.0) (National Biosciences). A second 5′ primer wasdesign to amplify the gene atpD for few enterobacteria difficult toamplify with the first primer set. When required, the primers containedinosines or degeneracies to account for variable positions.Oligonucleotide primers were synthesized with a model 394 DNA/RNAsynthesizer (PE Applied Biosystems). PCR primers used in this study arelisted in Table 20.

DNA Sequencing.

An 884-bp portion of the tuf gene and an 884-bp portion (oralternatively an 871-bp portion for a few enterobacterial strains) ofthe atpD gene were sequenced for all enterobacteria listed in the firststrain column of Table 19. Amplification was performed with 4 ng ofgenomic DNA. The 40-μ1 PCR mixtures used to generate PCR products forsequencing contained 1.0 μM each primer, 200 μM each deoxyribonucleosidetriphosphate (Pharmacia Biotech), 10 mM Tris-HCl (pH 9.0 at 25° C.), 50mM KCl, 0.1% (w/v) Triton X-100, 2.5 mM MgCl₂, 0.05 mM BSA, 0.3 U of TaqDNA polymerase (Promega) coupled with TaqStar™ antibody (ClontechLaboratories). The TaqStar™ neutralizing monoclonal antibody for Taq DNApolymerase was added to all PCR mixtures to enhance efficiency ofamplification (Kellogg et al., 1994). The PCR mixtures were subjected tothermal cycling (3 min at 95° C. and then 35 cycles of 1 min at 95° C.,1 min at 55° C. for tuf or 50° C. for atpD, and 1 min at 72° C., with a7-min final extension at 72° C.) using a PTC-200 DNA Engine thermocycler(MJ Research). PCR products having the predicted sizes were recoveredfrom an agarose gel stained for 15 min with 0.02% of methylene bluefollowed by washing in sterile distilled water for 15 min twice (Floreset al., 1992). Subsequently, PCR products having the predicted sizeswere recovered from gels using the QIAquick gel extraction kit (QIAGEN).

Both strands of the purified amplicons were sequenced using the ABIPrism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE AppliedBiosystems) on an automated DNA sequencer (Model 377). Amplicons fromtwo independent PCR amplifications were sequenced for each strain toensure the absence of sequencing errors attributable to nucleotidemiscorporations by the Taq DNA polymerase. Sequence assembly wasperformed with the aid of Sequencher 3.0 software (Gene Codes).

Phylogenetic Analysis.

Multiple sequence alignments were performed using PileUp from the GCGpackage (Version 10.0) (Genetics Computer Group) and checked by eye withthe editor SeqLab to edit sequences if necessary and to note whichregions were to be excluded for phylogenetic analysis. Vibrio choleraeand Shewanella putrefaciens were used as outgroups. Bootstrap subsets(750 sets) and phylogenetic trees were generated with the NeighborJoining algorithm from Dr. David Swofford's PAUP (Phylogenetic AnalysisUsing Parsimony) Software version 4.0b4 (Sinauer Associates) and withtree-bisection branch-swapping. The distance model used was Kimura(1980) two-parameter. Relative rate test was performed with the aid ofPhyltest program version 2.0 (c).

Results and Discussion

DNA Amplification, Sequencing And Sequence Alignments

A PCR product of the expected size of 884 bp was obtained for tuf and of884 or 871 bp for atpD from all bacterial strains tested. Aftersubtracting for biased primer regions and ambiguous single strand data,sequences of at least 721 bp for tuf and 713 bp for atpD were submittedto phylogenetic analyses. These sequences were aligned with tuf and atpDsequences available in databases to verify that the nucleotide sequencesindeed encoded a part of tested genes. Gaps were excluded to performphylogenetic analysis.

Signature Sequences

From the sequence alignments obtained from both tested genes, only oneinsertion was observed. This five amino acids insertion is locatedbetween the positions 325 and 326 of atpD gene of E. coli strain K-12(Saraste et al., 1981) and can be considered a signature sequence ofTatumella ptyseos and Pantoea species (FIG. 7). The presence of aconserved indel of defined length and sequence and flanked by conservedregions could suggest a common ancestor, particularly when members of agiven taxa share this indel (Gupta, 1998). To our knowledge, highrelatedness between the genera Tatumella and Pantoea is demonstrated forthe first time.

Enterobacter agglomerans ATCC 27989 sequence does not possess the fiveamino acid indel (FIG. 7). This indel could represent a useful marker tohelp resolve the Enterobacter agglomerans and Pantoea classification.Indeed, the transfer of Enterobacter agglomerans to Pantoea agglomeranswas proposed in 1989 by Gavini et al. (Gavini et al., 1989). However,some strains are provisionally classified as Pantoea sp. until theirinterrelatedness is elucidated (Gavini, et al., 1989). Since thetransfer was proposed, the change of nomenclature has not yet been madefor all Enterobacter agglomerans in the ATCC database. The absence ofthe five amino acids indel suggests that some strains of Enterobacteragglomerans most likely do not belong to the genus Pantoea.

Phylogenetic Trees Based on Partial Tuf Sequences, Atpd Sequences, andPublished 16S Rdna Data of Members of the Enterobacteriaceae.

Representative trees constructed from tuf and atpD sequences with theneighbor-joining method are shown in FIG. 8. The phylogenetic treesgenerated from partial tuf sequences and atpD sequences are verysimilar. Nevertheless, atpD tree shows more monophyletic groupscorresponding to species that belong to the same genus. These groups aremore consistent with the actual taxonomy. For both genes, some generaare not monophyletic. These results support previous phylogenies basedon the genes gap and ompA (Lawrence, et al., 1991), rpoB (Mollet, etal., 1997), and infB (Hedegaard, et al., 1999) which all showed that thegenera Escherichia and Klebsiella are polyphyletic. There were fewdifferences in branching between tuf and atpD genes.

Even though Pantoea agglomerans and Pantoea dispersa indels wereexcluded for phylogenetic analysis, these two species grouped togetherand were distant from Enterobacter agglomerans ATCC 27989, addinganother evidence that the latter species is heterogenous and that notall members of this species belong to the genus Pantoea. In fact, the E.agglomerans strain ATCC 27989 exhibits branch lengths similar to othersEnterobacter species with both genes. Therefore, we suggest that thisstrain belong to the genus Enterobacter until further reclassificationof that genus.

tuf and atpD trees exhibit very short genetic distances between taxabelonging to the same genetic species including species segregated forclinical considerations. This first concern E. coli and Shigella speciesthat were confirmed to be the same genetic species by hybridizationstudies (Brenner et al., 1972; Brenner et al., 1972; Brenner et al.,1982) and phylogenies based on 16S rDNA (Wang et al., 1997) and rpoBgenes (Mollet, et al., 1997). Hybridization studies (Bercovier, et al.,1980) and phylogeny based on 16S rDNA genes (Ibrahim et al., 1994)demonstrated also that Yersinia pestis and Y. pseudotuberculosis are thesame genetic species. Among Yersinia pestis and Y. pseudotuberculosis,the three Klebsiella pneumoniae subspecies, E. coli-Shigella species,and Salmonella choleraesuis subspecies, Salmonella is a less tightlyknit species than the other genetic species. The same is true for E.coli and Shigella species.

Escherichia fergusonii is very close to E. coli-Shigella geneticspecies. This observation is corroborated by 16S rDNA phylogeny(McLaughlin et al., 2000) but not by DNA hybridization values. In fact,E. fergusonii is only 49% to 63% related to E. coli-Shigella (FarmerIII, et al., 1985b). It was previously observed that very recentlydiverged species may not be recognizable based on 16S rDNA sequencesalthough DNA hybridization established them as different species (Fox etal., 1992). Therefore, E. fergusonii could be a new “quasi-species”.

atpD phylogeny revealed Salmonella subspecies divisions consistent withthe actual taxonomy. This result was already observed by Christensen etal. (Christensen & Olsen, 1998). Nevertheless, tuf partial sequencesdiscriminate less than atpD between Salmonella subspecies.

Overall, tuf and atpD phylogenies exhibit enough divergence betweenspecies to ensure efficient discrimination. Therefore, it could be easyto distinguish phenotypically close enterobacteria belonging todifferent genetic species such as Klebsiella pneumoniae and Enterobacteraerogenes.

Phylogenetic relationships between Salmonella, E. coli and C. freundiiare not well defined. 16S rDNA and 23S rDNA sequence data reveals acloser relationship between Salmonella and E. coli than betweenSalmonella and C. freundii (Christensen et al., 1998), while DNAhomology studies (Selander et al., 1996) and infB phylogeny (Hedegaard,et al., 1999) showed that Salmonella is more closely related to C.freundii than to E. coli. In that regard, tuf and atpD phylogenies arecoherent with 16S rDNA and 23S rDNA sequence analysis.

Phylogenetic analyses were also performed using amino acids sequences.tuf tree based on amino acids is characterized by a better resolutionbetween taxa outgroup and taxa ingroup (enterobacteria) than tree basedon nucleic acids whereas atpD trees based on amino acids and nucleicacids give almost the same resolution between taxa outgroup and ingroup(data not shown).

Relative rate test (or two cluster test (Takezaki et al., 1995))evaluates if evolution is constant between two taxa. Before to apply thetest, the topology of a tree is determined by tree-building methodwithout the assumption of rate constancy. Therefore, two taxa (or twogroups of taxa) are compared with a third taxon that is an outgroup ofthe first two taxa (Takezaki, et al., 1995). Few pairs of taxa thatexhibited a great difference between their branch lengths at particularnodes were chosen to perform the test. This test reveals that tuf andatpD are not constant in their evolution within the familyEnterobacteriaceae. For tuf, for example, the hypothesis of rateconstancy is rejected (Z value higher than 1.96) between Yersiniaspecies. The same is true for Proteus species. For atpD, for example,evolution is not constant between Proteus species, between Proteusspecies and Providencia species, and between Yersinia species andEscherichia coli. For 16S rDNA, for example, evolution is not constantbetween two E. coli, between E. coli and Enterobacter aerogenes, andbetween E. coli and Proteus vulgaris. These results suggest that tuf,atpD and 16S rDNA could not serve as a molecular clock for the entirefamily Enterobacteriaceae.

Since the number and the nature of taxa can influence topology of trees,phylogenetic trees from tuf and atpD were reconstructed using sequencescorresponding to strains for which 16S rDNA genes were published inGenEMBL. These trees were similar to those generated using 16S rDNA(FIGS. 9a, 9b and 9c ). Nevertheless, 16S rDNA tree gave poorerresolution power than tuf and atpD gene trees. Indeed, these latterexhibited less multifurcation (polytomy) than the 16S rDNA tree.

Comparison of Distances Based on tuf, atpD, and 16S rDNA Data.

tuf, atpD, and 16S rDNA distances (i.e. the number of differences pernucleotide site) were compared with each other for each pair of strains.We found that the tuf and atpD distances were respectively 2.268±0.965and 2.927±0.896 times larger than 16S rDNA distances (FIGS. 10a and b ).atpD distances were 1.445±0.570 times larger than tuf distances (FIG.10c ). FIGS. 10a, 10b and 10c also show that the tuf, atpD, and 16S rDNAdistances between members of different species of the same genus(0.053±0.034, 0.060±0.020, and 0.024±0.010, respectively) were in meansmaller than the distances between members of different genera belongingto the same family (0.103±0.053, 0.129±0.051, and 0.044±0.013,respectively). However, the overlap exhibits with standard deviationsadd to a focus of evidences that some enterobacterial genera are notwell defined (Brenner, 1984). In fact, many distances for pairs ofspecies especially belonging to the genera Escherichia, Shigella,Enterobacter, Citrobacter, Klebsiella, and Kluyvera overlap distancesfor pairs of species belonging to the same genus (FIGS. 10a, 10b and 10c). For example, distances for pairs composed by species of Citrobacterand species of Klebsiella overlap distances for pairs composed by twoCitrobacter or by two Klebsiella.

Observing the distance distributions, 16S rDNA distances reveal a clearseparation between the families Enterobacteriaceae and Vibrionaceaedespite the fact that the family Vibrionaceae is genetically very closeto the Enterobacteriaceae (FIGS. 10a and b ). Nevertheless, tuf and atpDshow higher discriminating power below the family level (FIGS. 10a and b).

There were some discrepancies in the relative distances for the samepairs of taxa between the two genes studied. First, distances betweenYersinia species are at least two times lower for atpD than for tuf(FIG. 10c ). Also, distances at the family level (betweenEnterobacteriaceae and Vibrionaceae) show that Enterobacteriaceae is atightlier knit family with atpD gene (Proteus genus excepted) than withtuf gene. Both genes well delineate taxa belonging to the same species.There is one exception with atpD: Klebsiella planticola and K.ornithinolithica belong to the same genus but fit with taxa belonging tothe same species (FIGS. 10a and c ). These two species are also veryclose genotypically with tuf gene. This suggest that Klebsiellaplanticola and K. ornithinolithica could be two newborn species. tuf andatpD genes exhibit little distances between Escherichia fergusonii andE. coli-Shigella species. Unfortunately, comparison with 16S rDNA couldnot be achieved because the E. fergusonii 16S rDNA sequence is not yetaccessible in GenEMBL database. Therefore, the majority ofphenotypically close enterobacteria could be easily discriminatedgenotypically using tuf and atpD gene sequences.

In conclusion, tuf and atpD genes exhibit phylogenies consistent with16S rDNA genes phylogeny. For example, they reveal that the familyEnterobacteriaceae is monophyletic. Moreover, tuf and atpD distancesprovide a higher discriminating power than 16S rDNA distances. In fact,tuf and atpD genes discriminate well between different genospecies andare conserved between strains of the same genetic species in such a waythat primers and molecular probes for diagnostic purposes could bedesigned. Preliminary studies support these observations and diagnostictests based on tuf and atpD sequence data to identify enterobacteria arecurrently under development.

Example 44

Testing New Pairs of PCR Primers Selected from Two Species-SpecificGenomic DNA Fragments which are Objects of Our Assigned U.S. Pat. No.6,001,564

Objective.

The goal of these experiments is to demonstrate that it is relativelyeasy for a person skilled in the art to find other PCR primer pairs fromthe species-specific fragments used as targets for detection andidentification of a variety of microorganisms. In fact, we wish to provethat the PCR primers previously tested by our group and which areobjects of the present patent application are not the only possible goodchoices for diagnostic purposes. For this example, we used diagnostictargets described in our assigned U.S. Pat. No. 6,001,564.

Experimental Strategy.

We have selected randomly two species-specific genomic DNA fragments forthis experiment. The first one is the 705-bp fragment specific toStaphylococcus epidermidis (SEQ ID NO: 36 from U.S. Pat. No. 6,001,564)while the second one is the 466-bp fragment specific to Moraxellacatarrhalis (SEQ ID NO: 29 from U.S. Pat. No. 6,001,564). Subsequently,we have selected from these two fragments a number of PCR primer pairsother than those previously tested. We have chosen 5 new primer pairsfrom each of these two sequences which are well dispersed along the DNAfragment (FIGS. 11 and 12). We have tested these primers for theirspecificity and compared them with the original primers previouslytested. For the specificity tests, we have tested all bacterial speciesclosely related to the target species based on phylogenetic analysiswith three conserved genes (rRNA genes, tuf and atpD). The rational forselecting a restricted number of bacterial species to evaluate thespecificity of the new primer pairs is based on the fact that the lackof specificity of a DNA-based assay is attributable to the detection ofclosely related species which are more similar at the nucleotide level.Based on the phylogenetic analysis, we have selected (i) species fromthe closely related genus Staphylococcus, Enterococcus, Streptococcusand Listeria to test the specificity of the S. epidermidis-specific PCRassays and (ii) species from the closely related genus Moraxella,Kingella and Neisseria to test the specificity of the M.catarrhalis-specific PCR assays.

Materials and Methods

Bacterial Strains.

All bacterial strains used for these experiments were obtained from theAmerican Type Culture Collection (ATCC, Rockville, Md.).

Genomic DNA Isolation.

Genomic DNA was purified from the ATCC reference strains by using theG-nome DNA kit (Bio 101 Inc., Vista, Calif.).

Oligonucleotide Design and Synthesis.

PCR primers were designed with the help of the Oligo™ primer analysissoftware Version 4.0 (National Biosciences Inc., Plymouth, Minn.) andsynthesized using a model 391 DNA synthesizer (Applied Biosystems,Foster City, Calif.).

PCR Assays.

All PCR assays were performed by using genomic DNA purified fromreference strains obtained from the ATCC. One μl of purified DNApreparation (containing 0.01 to 1 ng of DNA per μl) was added directlyinto the PCR reaction mixture. The 20 μL PCR reactions contained finalconcentrations of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100,2.5 mM MgCl₂, 0.4 μM of each primer, 200 μM of each of the four dNTPsand 0.5 unit of Taq DNA polymerase (Promega, Madison, Wis.) combinedwith the TaqStart™ antibody (Clontech Laboratories Inc., Palo Alto,Calif.). An internal control was integrated into all amplificationreactions to verify the efficiency of the amplification reaction as wellas to ensure that significant PCR inhibition was absent. Primersamplifying a region of 252 bp from a control plasmid added to eachamplification reaction were used to provide the internal control. PCRreactions were then subjected to thermal cycling (3 min at 95° C.followed by 30 cycles of 1 second at 95° C. for the denaturation stepand 30 seconds at 50 to 65° C. for the annealing-extension step) using aPTC-200 thermal cycler (MJ Research Inc., Watertown, Mass.). PCRamplification products were then analyzed by standard agarose gel (2%)electrophoresis. Amplification products were visualized in agarose gelscontaining 0.25 μg/mL of ethidium bromide under UV at 254 nm.

Results

Tables 21 and 22 show the results of specificity tests with the 5 newprimer pairs selected from SEQ ID NO: 29 (specific to M. catarrhalisfrom U.S. Pat. No. 6,001,564) and SEQ ID NO: 36 (specific to S.epidermidis from U.S. Pat. No. 6,001,564), respectively. In order toevaluate the performance of these new primers pairs, we compared them inparallel with the original primer pairs previously tested.

For M. catarrhalis, all of the 5 selected PCR primer pairs were specificfor the target species because none of the closely related species couldbe amplified (Table 21). In fact, the comparison with the originalprimer pair SEQ ID NO: 118+SEQ ID NO: 119 (from U.S. Pat. No. 6,001,564)revealed that all new pairs showed identical results in terms ofspecificity and sensitivity thereby suggesting their suitability fordiagnostic purposes.

For S. epidermidis, 4 of the 5 selected PCR primer pairs were specificfor the target species (Table 22). It should be noted that for 3 ofthese four primer pairs the annealing temperature had to be increasedfrom 55° C. to 60 or 65° C. to attain specificity for S. epidermidis.Again the comparison with the original primer pair SEQ ID NO: 145+SEQ IDNO: 146 (from U.S. Pat. No. 6,001,564) revealed that these four primerpairs were as good as the original pair. Increasing the annealingtemperature for the PCR amplification is well known by persons skilledin the art to be a very effective way to improve the specificity of aPCR assay (Persing et al., 1993, Diagnostic Molecular Microbiology:Principles and Applications, American Society for Microbiology,Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-based Diagnostics inInfectious Disease, Blackwell Scientific Publications, Boston, Mass.).In fact, those skilled in the art are well aware of the fact that theannealing temperature is critical for the optimization of PCR assays.Only the primer pair VBsep3+VBsep4 amplified bacterial species otherthan S. epidermidis including the staphylococcal species S. capitis, S.cohnii, S. aureus, S. haemolyticus and S. hominis (Table 22). For thisnon-specific primer pair, increasing the annealing temperature from 55to 65° C. was not sufficient to attain the desired specificity. Onepossible explanation for the fact that it appears slightly easier toselect species-specific primers for M. catarrhalis than for S.epidermidis is that M. catarrhalis is more isolated in phylogenetictrees than S. epidermidis. The large number of coagulase negativestaphylococcal species such as S. epidermidis is largely responsible forthis phylogenetic clustering.

Conclusion

These experiment clearly show that it is relatively easy for a personskilled in the art to select, from the species-specific DNA fragmentsselected as target for identification, PCR primer pairs suitable fordiagnostic purposes other than those previously tested. Theamplification conditions can be optimize by modifying critical variablessuch as the annealing temperature to attain the desired specificity andsensitivity. Consequently, we consider that it is legitimate to claimany possible primer sequences selected from the species-specificfragment and that it would be unfair to grant only the claims dealingwith the primer pairs previously tested. By extrapolation, these resultsstrongly suggest that it is also relatively easy for a person skilled inthe art to select, from the species-specific DNA fragments, DNA probessuitable for diagnostic purposes other than those previously tested.

Example 45

Testing Modified Versions of PCR Primers Derived from the Sequence ofSeveral Primers which are objects of U.S. Pat. No. 6,001,564.

Objective.

The purpose of this project is to verify the efficiency of amplificationby modified PCR primers derived from primers previously tested. Thetypes of primer modifications to be tested include (i) variation of thesequence at one or more nucleotide positions and (ii) increasing orreducing the length of the primers. For this example, we used diagnostictargets described in U.S. Pat. No. 6,001,564.

Experimental Strategy:

Testing Primers with Nucleotide Changes

We have designed 13 new primers which are derived from the S.epidermidis-specific SEQ ID NO: 146 from U.S. Pat. No. 6,001,564 (Table23). These primers have been modified at one or more nucleotidepositions. As shown in Table 23, the nucleotide changes were introducedall along the primer sequence. Furthermore, instead of modifying theprimer at any nucleotide position, the nucleotide changes wereintroduced at the third position of each codon to better reflectpotential genetic variations in vivo. It should be noted that nonucleotide changes were introduced at the 3′ end of the oligonucleotideprimers because those skilled in the art are well aware of the fact thatmimatches at the 3′ end should be avoided (Persing et al., 1993,Diagnostic Molecular Microbiology: Principles and Applications, AmericanSociety for Microbiology, Washington, D.C.). All of these modifiedprimers were tested in PCR assays in combination with SEQ ID NO: 145from U.S. Pat. No. 6,001,564 and the efficiency of the amplification wascompared with the original primer pair SEQ ID NO: 145+SEQ ID NO: 146previously tested in U.S. Pat. No. 6,001,564.

Testing Shorter or Longer Versions of Primers

We have designed shorter and longer versions of the original S.epidermidis-specific PCR primer pair SEQ ID NO: 145+146 from U.S. Pat.No. 6,001,564 (Table 24) as well as shorter versions of the original P.aeruginosa-specific primer pair SEQ ID NO: 83+84 from U.S. Pat. No.6,001,564 (Table 25). As shown in Tables 24 and 25, both primers of eachpair were shortened or lengthen to the same length. Again, those skilledin the art know that the melting temperature of both primers from a pairshould be similar to avoid preferential binding at one primer bindingsite which is detrimental in PCR (Persing et al., 1993, DiagnosticMolecular Microbiology: Principles and Applications, American Societyfor Microbiology, Washington, D.C.; Ehrlich and Greenberg, 1994,PCR-based Diagnostics in Infectious Disease, Blackwell ScientificPublications, Boston, Mass.). All of these shorter or longer primerversions were tested in PCR assays and the efficiency of theamplification was compared with the original primer pair SEQ ID NOs 145and 146.

Materials and Methods

See the Materials and methods section of Example 44.

Results

Testing Primers with Nucleotide Changes

The results of the PCR assays with the 13 modified versions of SEQ IDNO: 146 from U.S. Pat. No. 6,001,564 are shown in Table 23. The 8modified primers having a single nucleotide variation showed anefficiency of amplification identical to the original primer pair basedon testing with 3 different dilutions of genomic DNA. The four primershaving two nucleotide variations and primer VBmut12 having 3 nucleotidechanges also showed PCR results identical to those obtained with theoriginal pair. Finally, primer VBmut13 with four nucleotide changesshowed a reduction in sensitivity by approximately one log as comparedwith the original primer pair. However, reducing the annealingtemperature from 55 to 50° C. gave an efficiency of amplification verysimilar to that observed with the original primer pair (Table 23). Infact, reducing the annealing temperature of PCR cycles represents aneffective way to reduce the stringency of hybridization for the primersand consequently allows the binding of probes with mismatches (Persinget al., 1993, Diagnostic Molecular Microbiology: Principles andApplications, American Society for Microbiology, Washington, D.C.).Subsequently, we have confirmed the specificity of the PCR assays witheach of these 13 modified versions of SEQ ID NO: 146 from U.S. Pat. No.6,001,564 by performing amplifications from all bacterial speciesclosely related to S. epidermidis which are listed in Table 22.

Testing Shorter or Longer Versions of Primers

For these experiments, two primer pairs were selected: i) SEQ ID NO:145+146 from U.S. Pat. No. 6,001,564 (specific to S. epidermidis) whichare AT rich and ii) SEQ ID NO: 83+84 (specific to P. aeruginosa) whichare GC rich. For the AT rich sequence, primers of 15 to 30 nucleotide inlength were designed (Table 24) while for the GC rich sequences, primersof 13 to 19 nucleotide in length were designed (Table 25).

Table 24 shows that, for an annealing temperature of 55° C., the 30-25-,20- and 17-nucleotide versions of SEQ ID NO: 145 and 146 from U.S. Pat.No. 6,001,564 all showed identical results as compared with the originalprimer pair except that the 17-nucleotide version amplified slightlyless efficiently the S. epidermidis DNA. Reducing the annealingtemperature from 55 to 45° C. for the 17-nucleotide version allowed toincrease the amplification efficiency to a level very similar to thatwith the original primer pair (SEQ ID NO: 145+146 from U.S. Pat. No.6,001,564). Regarding the 15-nucleotide version, there was amplificationof S. epidermidis DNA only when the annealing temperature was reduced to45° C. Under those PCR conditions the assay remained S.epidermidis-specific but the amplification signal with S. epidermidisDNA was slightly lower as compared with the original primer pair.Subsequently, we have further confirmed the specificity of the shorteror longer versions by amplifying DNA from all bacterial species closelyrelated to S. epidermidis which are listed in Table 22.

Table 25 shows that, for an annealing temperature of 55° C., all shorterversions of SEQ ID NO: 83 and 84 from U.S. Pat. No. 6,001,564 showedidentical PCR results as compared with the original primer pair. Asexpected, these results show that it is simpler to reduce the length ofGC rich as compared with AT rich. This is attributable to the fact thatGC binding is more stable than AT binding.

Conclusion

Testing Primers with Nucleotide Changes

The above experiments clearly show that PCR primers may be modified atone or more nucleotide positions without affecting the specificity andthe sensitivity of the PCR assay. These results strongly suggest that agiven oligonucleotide can detect variant genomic sequences from thetarget species. In fact, the nucleotide changes in the selected primerswere purposely introduced at the third position of each codon to mimicnucleotide variation in genomic DNA. Thus we conclude that it isjustified to claim “a variant thereof” for i) the SEQ IDs of thefragments and oligonucleotides which are object of the present patentapplication and ii) genomic variants of the target species.

Testing Shorter or Longer Versions of Primers

The above experiments clearly show that PCR primers may be shorter orlonger without affecting the specificity and the sensitivity of the PCRassay. We have showed that oligonucleotides ranging in sizes from 13 to30 nucleotides may be as specific and sensitive as the original primerpair from which they were derived. Consequently, these results suggestthat it is not exaggerated to claim sequences having at least 12nucleotide in length.

This invention has been described herein above, and it is readilyapparent that modifications can be made thereto without departing fromthe spirit of this invention. These modifications are under the scope ofthis invention, as defined in the appended claims.

TABLE 1 Distribution (%) of nosocomial pathogens for various humaninfections in USA (1990-1992)¹. Pathogen UTI² SSI³ BSI⁴ Pneumonia CSF⁵Escherichia coli 27 9 5 4 2 Staphylococcus aureus 2 21 17 21 2Staphylococcus epidermidis 2 6 20 0 1 Enterococcus faecalis 16 12 9 2 0Enterococcus faecium 1 1 0 0 0 Pseudomonas aeruginosa 12 9 3 18 0Klebsiella pneumoniae 7 3 4 9 0 Proteus mirabilis 5 3 1 2 0Streptococcus pneumoniae 0 0 3 1 18 Group B Streptococci 1 1 2 1 6 Otherstreptococci 3 5 2 1 3 Haemophilus influenzae 0 0 0 6 45 Neisseriameningitidis 0 0 0 0 14 Listeria monocytogenes 0 0 0 0 3 Otherenterococci 1 1 0 0 0 Other staphylococci 2 8 13 2 0 Candida albicans 93 5 5 0 Other Candida 2 1 3 1 0 Enterobacter sp. 5 7 4 12 2Acinetobacter sp. 1 1 2 4 2 Citrobacter sp. 2 1 1 1 0 Serratiamarcescens 1 1 1 3 1 Other Klebsiella 1 1 1 2 1 Others 0 6 4 5 0 ¹Datarecorded by the National Nosocomial Infections Surveillance (NNIS) from80 hospitals (Emori and Gaynes, 1993, Clin. Microbiol. Rev., 6:428-442). ²Urinary tract infection. ³Surgical site infection.⁴Bloodstream infection. ⁵Cerebrospinal fluid.

TABLE 2 Distribution (%) of bloodstream infection pathogens in Quebec(1995), Canada (1992), UK (1969-1988) and USA (1990-1992). UK³ USA⁴Community- Hospital- Hospital- Organism Quebec¹ Canada² acquiredacquired acquired E. coli 15.6  53.8  24.8  20.3  5.0 S. epidermidis25.8  — 0.5 7.2 31.0  and other CoNS⁵ S. aureus 9.6 — 9.7 19.4  16.0  S.pneumoniae 6.3 — 22.5  2.2 — E. faecalis 3.0 — 1.0 4.2 — E. faecium 2.6— 0.2 0.5 — Enterococcus sp. — — — — 9.0 H. influenzae 1.5 — 3.4 0.4 —P. aeruginosa 1.5 8.2 1.0 8.2 3.0 K. pneumoniae 3.0 11.2  3.0 9.2 4.0 P.mirabilis — 3.9 2.8 5.3 1.0 S. pyogenes — — 1.9 0.9 — Enterobacter sp.4.1 5.5 0.5 2.3 4.0 Candida sp. 8.5 — — 1.0 8.0 Others 18.5  17.4  28.7 18.9  19.0  ¹Data obtained for 270 isolates collected at the CentreHospitalier de l'Université Laval (CHUL) during a 5 month period (May toOctober 1995). ²Data from 10 hospitals throughout Canada representing941 gram-negative isolates. (Chamberland et al., 1992, Clin. Infect.Dis., 15: 615-628). ³Data from a 20-year study (1969-1988) for nearly4000 isolates. (Eykyn et al., 1990, J. Antimicrob. Chemother., Suppl. C,25: 41-58). ⁴Data recorded by the National Nosocomial InfectionsSurveillance (NNIS) from 80 hospitals (Emori and Gaynes, 1993, Clin.Microbiol. Rev., 6: 428-442). ⁵Coagulase-negative staphylococci.

TABLE 3 Distribution of positive and negative clinical specimens testedat the microbiology laboratory of the CHUL (February 1994-January 1995).% of % of Clinical specimens No. of samples positive negative and/orsites tested (%) specimens specimens Urine 17,981 (54.5) 19.4 80.6 Bloodculture/marrow 10,010 (30.4) 6.9 93.1 Sputum 1,266 (3.8) 68.4 31.6Superficial pus 1,136 (3.5) 72.3 27.7 Cerebrospinal fluid 553 (1.7) 1.099.0 Synovial fluid 523 (1.6) 2.7 97.3 Respiratory tract 502 (1.5) 56.643.4 Deep pus 473 (1.4) 56.8 43.2 Ears 289 (0.9) 47.1 52.9 Pleural andpericardial fluid 132 (0.4) 1.0 99.0 Peritoneal fluid 101 (0.3) 28.671.4 Total: 32,966 (100.0) 20.0 80.0

Table 4 Example of microbial species for which tuf and/or atpD and/orrecA nucleic acids and/or sequences are used in the present invention.Bacterial species Abiotrophia adiacens Abiotrophia defectivaAchromobacter xylosoxidans subsp. denitrificans Acetobacterium woodiAcetobacter aceti Acetobacter altoacetigenes Acetobacter polyoxogenesAcholeplasma laidlawii Acidothermus cellulolyticus Acidiphilum facilisAcinetobacter baumannii Acinetobacter calcoaceticus Acinetobacterlwoffii Actinomyces meyeri Aerococcus viridans Aeromonas hydrophilaAeromonas salmonicida Agrobacterium radiobacter Agrobacteriumtumefaciens Alcaligenes faecalis subsp. faecalis Allochromatium vinosumAnabaena variabilis Anacystis nidulans Anaerorhabdus furcosus Aquifexaeolicus Aquifex pyrophilus Arcanobacterium haemolyticum Archaeoglobusfulgidus Azotobacter vinelandii Bacillus anthracis Bacillus cereusBacillus firmus Bacillus halodurans Bacillus megaterium Bacillusmycoides Bacillus pseudomycoides Bacillus stearothermophilus Bacillussubtilis Bacillus thuringiensis Bacillus weihenstephanensis Bacteroidesdistasonis Bacteroides fragilis Bacteroides forsythus Bacteroides ovatusBacteroides vulgatus Bartonella henselae Bifidobacterium adolescentisBifidobacterium breve Bifidobacterium dentium Bifidobacterium longumBlastochloris viridis Borrelia burgdorferi Bordetella pertussisBordetella bronchi septica Brucella abortus Brevibacterium linensBrevibacterium flavum Brevundimonas dim inuta Buchnera aphidicolaBudvicia aquatica Burkholderia cepacia Burkholderia mallei Burkholderiapseudomallei Buttiauxella agrestis Butyrivibrio fibrisolvensCampylobacter coli Campylobacter curvus Campylobacter fetus subsp. fetusCampylobacter fetus subsp. venerealis Campylobacter gracilisCampylobacter jejuni Campylobacter jejuni subsp. doylei Campylobacterjejuni subsp. jejuni Campylobacter lari Campylobacter rectusCampylobacter sputorum subsp. sputorum Campylobacter upsaliensis Cedeceadavisae Cedecea lapagei Cedecea neteri Chlamydia pneumoniae Chlamydiapsittaci Chlamydia trachomatis Chlorobium vibrioforme Chlorollexusaurantiacus Chryseobacterium meningosepticum Citrobacter amalonaticusCitrobacter braakii Citrobacter farmeri Citrobacter freundii Citrobacterkoseri Citrobacter sedlakii Citrobacter werkmanii Citrobacter youngaeClostridium acetobutylicum Clostridium beijerinckii Clostridiumbifermentans Clostridium botulinum Clostridium difficile Clostridiuminnocuum Clostridium histolyticum Clostridium novyi Clostridiumperfringens Clostridium ramosum Clostridium septicum Clostridiumsordellii Clostridium tertium Clostridium tetani Comamonas acidovoransCorynebacterium accolens Corynebacterium bovis Corynebacterium cervicisCorynebacterium diphtheriae Corynebacterium flavescens Corynebacteriumgenitalium Corynebacterium glutamicum Corynebacterium jeikeiumCorynebacterium kutscheri Corynebacterium minutissimum Corynebacteriummycetoides Corynebacterium pseudodiphtheriticum Corynebacteriumpseudogenitalium Corynebacterium pseudotuberculosis Corynebacteriumrenale Corynebacterium striatum Corynebacterium ulcerans Corynebacteriumurealyticum Corynebacterium xerosis Coxiella bumetii Cytophaga lyticaDeinococcus radiodurans Deinonema sp. Edwardsiella hoshinae Edwardsiellatarda Ehrlichia canis Ehrlichia risticii Eikenella corrodensEnterobacter aerogenes Enterobacter agglomerans Enterobacter amnigenusEnterobacter asburiae Enterobacter cancerogenus Enterobacter cloacaeEnterobacter gergoviae Enterobacter hormaechei Enterobacter sakazakiiEnterococcus avium Enterococcus casseliflavus Enterococcus cecorumEnterococcus columbae Enterococcus dispar Enterococcus duransEnterococcus faecalis Enterococcus faecium Enterococcus flavescensEnterococcus gallinarum Enterococcus hirae Enterococcus malodoratusEnterococcus mundtii Enterococcus pseudoavium Enterococcus raffinosusEnterococcus saccharolyticus Enterococcus solitarius Enterococcussulfureus Erwinia amylovora Erwinia carotovora Escherichia coliEscherichia fergusonii Escherichia hermannii Escherichia vulnerisEubacterium lentum Eubacterium nodatum Ewingella americana Francisellatularensis Frankia alni Fervidobacterium islandicum Fibrobactersuccinogenes Flavobacterium ferrigeneum Flexistipes sinusarabiciFusobacterium gonidiaformans Fusobacterium necrophorum subsp.necrophorum Fusobacterium nucleatum subsp. polymorphum Gardnerellavaginalis Gemella haemolysans Gemella morbillorum Globicatella sanguisGloeobacter violaceus Gloeothece sp. Gluconobacter oxydans Haemophilusactinomycetemcomitans Haemophilus aphrophilus Haemophilus ducreyiHaemophilus haemolyticus Haemophilus influenzae Haemophilusparahaemolyticus Haemophilus parainfluenzae Haemophilus paraphrophilusHaemophilus segnis Hafnia alvei Halobacterium marismortui Halobacteriumsalinarum Haloferax volcanii Helicobacter pylori Herpetoshiphonaurantiacus Kingella kingae Klebsiella omithinolytica Klebsiella oxytocaKlebsiella planticola Klebsiella pneumoniae subsp. ozaenae Klebsiellapneumoniae subsp. pneumoniae Klebsiella pneumoniae subsp.rhinoscleromatis Klebsiella terrigena Kluyvera ascorbata Kluyveracryocrescens Kluyvera georgiana Kocuria kristinae Lactobacillusacidophilus Lactobacillus garvieae Lactobacillus paracasei Lactobacilluscasei subsp. casei Legionella micdadei Legionella pneumophila subsp.pneumophila Leminorella grimontii Leminorella richardii Leptospirabillexa Leptospira interrogans Leuconostoc mesenteroides subsp.dextranicum Listeria innocua Listeria ivanovii Listeria monocytogenesListeria seeligeri Macrococcus caseolyticus Magnetospirillummagnetotacticum Megamonas hypermegale Methanobacteriumthermoautotrophicum Methanococcus jannaschii Methanococcus vannieliiMethanosarcina barkeri Methanosarcina jannaschii Methylobacillusflagellatum Methylomonas clara Micrococcus luteus Micrococcus lylaeMitsuokella multacidus Mobiluncus curtisii subsp. holmesii Moellerellathermoacetica Moellerella wisconsensis Moorella therm oacetica Moraxellacatarrhalis Moraxella osloensis Morganella morganii subsp. morganiiMycobacterium avium Mycobacterium bovis Mycobacterium gordonaeMycobacterium kansasii Mycobacterium leprae Mycobacterium terraeMycobacterium tuberculosis Mycoplasma capricolum Mycoplasmagallisepticum Mycoplasma genitalium Mycoplasma hominis Mycoplasma pirumMycoplasma mycoides Mycoplasma pneumoniae Mycoplasma pulmonis Mycoplasmasalivarium Myxococcus xanthus Neisseria animalis Neisseria canisNeisseria cinerea Neisseria cuniculi Neisseria elongata subsp. elongataNeisseria elongata subsp. intermedia Lactococcus garvieae Lactococcuslactis Lactococcus lactis subsp. lactis Leclercia adecarboxylataNeisseria flava Neisseria flavescens Neisseria gonorrhoeae Neisserialactamica Neisseria meningitidis Neisseria mucosa Neisseria perflavaNeisseria pharyngis var. flava Neisseria polysaccharea Neisseria siccaNeisseria subflava Neisseria weaveri Obesum bacterium proteusOchrobactrum anthropi Pantoea agglomerans Pantoea dispersa Paracoccusdenitrifi cans Pasteurella multocida Pectinatus frisingensis Peptococcusniger Peptostreptococcus anaerobius Peptostreptococcus asaccharolyticusPeptostreptococcus prevotii Phormidium ectocarpi Pirellula marinaPlanobispora rosea Plesiomonas shigelloides Plectonema boryanumPorphyromonas asaccharolytica Porphyromonas gingiva/is Pragia fontiumPrevotella buccalis Prevotella melaninogenica Prevotella oralisPrevotella ruminocola Prochlorothrix hollandica Propionibacterium acnesPropionigenium modestum Proteus mirabilis Proteus penneri Proteusvulgaris Providencia alcalifaciens Providencia rettgeri Providenciarustigianii Providencia stuartii Pseudomonas aeruginosa Pseudomonasfluorescens Pseudomonas putida Pseudomonas stutzeri Psychrobacterphenylpyruvicum Pyrococcus abyssi Rahnella aquatilis Rickettsiaprowazekii Rhizobium leguminosarum Rhizobium phaseoli Rhodobactercapsulatus Rhodobacter sphaeroides Rhodopseudomonas palustrisRhodospirillum rubrum Ruminococcus albus Ruminococcus bromii Salmonellabongori Salmonella choleraesuis subsp. arizonae Salmonella choleraesuissubsp choleraesuis Salmonella choleraesuis subsp. diarizonae Salmonellacholeraesuis subsp. houtenae Salmonella choleraesuis subsp. indicaSalmonella choleraesuis subsp. salamae Serpulina hyodysenteriae Serratiaficaria Serratia fonticola Serratia grimesii Serratia liquefaciensSerratia marcescens Serratia odorifera Serratia plymuthica Serratiarubidaea Shewanella putrefaciens Shigella boydii Shigella dysenteriaeShigella flexneri Shigella sonnei Sinorhizobium meliloti Spirochaetaaurantia Staphylococcus aureus Staphylococcus aureus subsp. aureusStaphylococcus auricularis Staphylococcus capitis subsp. capitisStaphylococcus cohnii subsp. cohnii Staphylococcus epidermidisStaphylococcus haemolyticus Staphylococcus hominis Staphylococcushominis subsp. hominis Staphylococcus lugdunensis Staphylococcussaprophyticus Staphylococcus sciuri subsp. sciuri Staphylococcussimulans Staphylococcus warneri Stigmatella aurantiaca Stenotrophomonasmaltophilia Streptococcus acidominimus Streptococcus agalactiaeStreptococcus anginosus Streptococcus bovis Streptococcus cricetusStreptococcus cri status Streptococcus downei Streptococcus dysgalactiaeStreptococcus equi subsp. equi Streptococcus ferus Streptococcusgordonii Streptococcus macacae Streptococcus mitis Streptococcus mutansStreptococcus oralis Streptococcus parasanguinis Streptococcuspneumoniae Streptococcus pyogenes Streptococcus ratti Streptococcussalivarius Streptococcus salivarius subsp. thermophilus Streptococcussanguinis Streptococcus sobrinus Streptococcus suis Streptococcus uberisStreptococcus vestibularis Streptomyces anbofaciens Streptomycesaureofaciens Streptomyces cinnamoneus Streptomyces coelicolorStreptomyces collinus Streptomyces lividans Streptomyces netropsisStreptomyces ramocissimus Streptomyces rimosus Streptomyces venezuelaeSuccinivibrio dextrinosolvens Synechococcus sp. Synechocystis sp.Tatumella ptyseos Taxeobacter occealus Tetragenococcus halophilusThermoplasma acidophilum Therm otoga maritima Therms aquaticus Thermsthermophilus Thiobacillus ferrooxidans Thiomonas cuprina Trabulsiellaguamensis Treponema pallidum Ureaplasma urealyticum Veillonella parvulaVibrio alginolyticus Vibrio anguillarum Vibrio cholerae Vibrio mimicusWohnella succinogenes Xanthomonas citri Xanthomonas oryzae Xenorhabdusbovieni Xenorhabdus nematophilus Yersinia bercovieri Yersiniaenterocolitica Yersinia frederiksensii Yersinia intermedia Yersiniapestis Yersinia pseudotuberculosis Yersinia rohdei Yokenellaregensburgei Zoogloea ramigera Fungal species Absidia corymbiferaAbsidia glauca Alternaria alternata Arxula adeninivorans Aspergillusflavus Aspergillus fumigatus Aspergillus nidulans Aspergillus nigerAspergillus oryzae Aspergillus terreus Aspergillus versicolorAureobasidium pullulans Basidiobolus ranarum Bipolaris hawaiiensisBilophila wadsworthia Blastoschizomyces capitatus Blastomycesdermatitidis Candida albicans Candida catenulata Candida dubliniensisCandida famata Candida glabrata Candida guilliermondii Candidahaemulonii Candida inconspicua Candida kefyr Candida krusei Candidalambica Candida lusitaniae Candida norvegica Candida norvegensis Candidaparapsilosis Candida rugosa Candida sphaerica Candida tropicalis Candidautilis Candida viswanathii Candida zeylanoides Cladophialophoracarrionii Coccidioides immitis Coprinus cinereus Cryptococcus albidusCryptococcus humicolus Cryptococcus laurentii Cryptococcus neoformansCunninghamella bertholletiae Curvularia lunata Emericella nidulansEmmonsia parva Eremothecium gossypii Exophiala dermatitidis Exophialajeanselmei Exophiala moniliae Exserohilum rostratum Eremotheciumgossypii Fonsecaea pedrosoi Fusarium moniliforme Fusarium oxysporumFusarium solani Geotrichum sp. Histoplasma capsulatum Hortaea werneckiiIssatchenkia orientalis Kudrjanzev Kluyveromyces lactis Malasseziafurfur Malassezia pachydermatis Malbranchea filamentosa Metschnikowiapulcherrima Microsporum audouinii Microsporum canis Mucor circinelloidesNeurospora crassa Paecilomyces lilacinus Paracoccidioides brasiliensisPenicillium marneffei Phialaphora verrucosa Pichia anomala Piedraiahortai Podospora anserina Podospora curvicolla Puccinia graminisPseudallescheria boydii Reclinomonas americana Rhizomucor racemosusRhizopus oryzae Rhodotorula minuta Rhodotorula mucilaginosaSaccharomyces cerevisiae Saksenaea vasiformis Schizosaccharomyces pombeScopulariopsis koningii Sordaria macrospora Sporobolomyces salmonicolorSporothrix schenckii Stephanoascus ciferrii Syncephalastrum racemosumTrichoderma reesei Trichophyton mentagrophytes Trichophyton rubrumTrichophyton tonsurans Trichosporon cutaneum Ustilago maydis Wangielladermatitidis Yarrowia lipolytica Parasitical species Babesia bigeminaBabesia bovis Babesia micron Blastocystis hominis Crithidia fasciculataCryptosporidium parvum Entamoeba histolytica Giardia lambliaKentrophoros sp. Leishmania aethiopica Leishmania amazonensis Leishmaniabraziliensis Leishmania donovani Leishmania infantum Leishmaniaenriettii Leishmania gerbilli Leishmania guyanensis Leishmania hertigiLeishmania major Leishmania mexicana Leishmania panamensis Leishmaniatarentolae Leishmania tropica Neospora caninum Onchocerca volvulusPlasmodium berghei Plasmodium falciparum Plasmodium knowlesi Porphyrapurpurea Toxoplasma gondii Treponema pallidum Trichomonas tenaxTrichomonas vaginalis Trypanosoma brucei Trypanosoma brucei subsp.brucei Trypanosoma con golense Trypanosoma cruzi

TABLE 5 Antimicrobial agents resistance genes selected for diagnosticpurposes ACCES- SEQ Gene Antimicrobial agent Bacteria¹ SION NO. ID NO.aac(3)-Ib² Aminoglycosides Enterobacteriaceae L06157 Pseudomonadsaac(3)-IIb² Aminoglycosides Enterobacteriaceae, M97172 Pseudomonadsaac(3)-IVa² Aminoglycosides Enterobacteriaceae X01385 aac(3)-VIa²Aminoglycosides Enterobacteriaceae, M88012 Pseudomonads aac(2′)-1a²Aminoglycosides Enterobacteriaceae, X04555 Pseudomonads aac(6′)-aph(2″)²Aminoglycosides Enterococcus sp., 83-86³ Staphylococcus sp. aac(6′)-Ia,²Aminoglycosides Enterobacteriaceae, M18967 Pseudomonads aac(6′)-Ic²Aminoglycosides Enterobacteriaceae, M94066 Pseudomonads aac(6′)-IIa²Aminoglycosides Pseudomonads 112⁴ aadB [ant(2″)-Ia²] AminoglycosidesEnterobacteriaceae 53-54³ aacC1 [aac(3)-Ia²] AminoglycosidesPseudomonads 55-56³ aacC2 [aac(3)-IIa²] Aminoglycosides Pseudomonads57-58³ aacC3 [aac(3)-III²] Aminoglycosides Pseudomonads 59-60³ aacA4[aac(6′)-Ib²] Aminoglycosides Pseudomonads 65-66³ ant(3″)-Ia²Aminoglycosides Enterobacteriaceae, X02340 Enterococcus sp., M10241Staphylococcus sp. ant(4′)-Ia² Aminoglycosides Staphylococcus sp. V01282aph(3′)-Ia² Aminoglycosides Enterobacteriaceae, J01839 Pseudomonadsaph(3′)-IIa² Aminoglycosides Enterobacteriaceae, V00618 Pseudomonadsaph(3′)-IIIa² Aminoglycosides Enterococcus sp., V01547 Staphylococcussp. aph(3′)-VIa² Aminoglycosides Enterobacteriaceae, X07753 PseudomonadsrpsL² Streptomycin M. tuberculosis, X80120 M. avium complex U14749X70995 L08011 bla_(OXA) ^(5,6) β-lactams Enterobacteriaceae, Y10693 110⁴Pseudomonads AJ238349 AJ009819 X06046 X03037 X07260 U13880 X75562AF034958 J03427 Z22590 U59183 L38523 U63835 AF043100 AF060206 U85514AF043381 AF024602 AF064820 bla_(ROB) ⁵ β-lactams Haemophilus sp. 45-48³bla_(SHV) ^(5,6) β-lactams Enterobacteriacea, AF124984 41-44³Pseudomonas aeruginosa AF148850 M59181 X98099 M33655 AF148851 X53433L47119 AF074954 X53817 AF096930 X55640 Y11069 U20270 U92041 S82452X98101 X98105 AF164577 AJ011428 AF116855 AB023477 AF293345 AF227204AF208796 AF132290 bla_(TEM) ^(5,6) β-lactams Enterobacteriaceae,AF012911 37-40³ Neisseria sp., U48775 Haemophilus sp. AF093512 AF052748X64523 Y13612 X57972 AF157413 U31280 U36911 U48775 V00613 X97254AJ012256 X04515 AF126482 U09188 M88143 Y14574 AF188200 AJ251946 Y17581Y17582 Y17583 M88143 U37195 Y17584 X64523 U95363 Y10279 Y10280 Y10281AF027199 AF104441 AF104442 AF062386 X57972 AF047171 AF188199 AF157553AF190694 AF190695 AF190693 AF190692 bla_(SHV) ^(5,6) β-lactamsEnterobacteriacea, AF124984 41-44³ Pseudomonas aeruginosa AF148850M59181 X98099 M33655 AF148851 X53433 L47119 AF074954 X53817 AF096930X55640 Y11069 U20270 U92041 S82452 X98101 X98105 AF164577 AJ011428AF116855 AB023477 AF293345 AF227204 AF208796 AF132290 bla_(TEM) ^(5,6)β-lactams Enterobacteriaceae, AF012911 37-40³ Neisseria sp., U48775Haemophilus sp. AF093512 AF052748 X64523 Y13612 X57972 AF157413 U31280U36911 U48775 V00613 X97254 AJ012256 X04515 AF126482 U09188 M88143Y14574 AF188200 AJ251946 Y17581 Y17582 Y17583 M88143 U37195 Y17584X64523 U95363 Y10279 Y10280 Y10281 AF027199 AF104441 AF104442 AF062386X57972 AF047171 AF188199 AF157553 AF190694 AF190695 AF190693 AF190692bla_(CARB) ⁵ β-lactams Pseudomonas sp., J05162 Enterobacteriaceae S46063M69058 U14749 D86225 D13210 Z18955 AF071555 AF153200 AF030945bla_(CTX-M-1) ⁵ β-lactams Enterobacteriaceae X92506 bla_(CTX-M-2) ⁵β-lactams Enterobacteriaceae X92507 bla_(CMY-2) ⁷ β-lactamsEnterobacteriaceae X91840 AJ007826 AJ011293 AJ011291 Y17716 Y16783Y16781 Y15130 U77414 S83226 Y15412 X78117 bla_(IMP) ⁵ β-lactamsEnterobacteriaceae, AJ223604 Pseudomonas aeruginosa S71932 D50438 D29636X98393 AB010417 D78375 bla_(PER-1) ⁵ β-lactams Enterobacteriaceae,Z21957 Pseudomodanaceae bla_(PER-2) ⁷ β-lactams EnterobacteriaceaeX93314 blaZ¹² β-lactams Enterococcus sp., 111⁴ Staphylococcus sp. mecA¹²β-lactams Staphylococcus sp. 97-98³ pbp1a¹³ β-lactams Streptococcuspneumoniae M90527 1004-1018, X67872 1648, 2056-2064, AB006868 2273-2276AB006874 X67873 AB006878 AB006875 AB006877 AB006879 AF046237 AF046235AF026431 AF046232 AF046233 AF046236 X67871 Z49095 AF046234 AB006873X67866 X67868 AB006870 AB006869 AB006872 X67870 AB006871 X67867 X67869AB006876 AF046230 AF046238 Z49094 pbp2b¹³ β-lactams Streptococcuspneumoniae X16022 1019-1033  M25516 M25518 M25515 U20071 U20084 U20082U20067 U20079 Z22185 U20072 pbp2b¹³ β-lactams Streptococcus pneumoniaeU20083 U20081 M25522 U20075 U20070 U20077 U20068 Z22184 U20069 U20078M25521 M25525 M25519 Z21981 M25523 M25526 U20076 U20074 M25520 M25517M25524 Z22230 U20073 U20080 pbp2x¹³ β-lactams Streptococcus pneumoniaeX16367 1034-1048  X65135 AB011204 AB011209 AB011199 AB011200 AB011201AB011202 AB011198 AB011208 AB011205 AB015852 AB011210 AB015849 AB015850AB015851 AB015847 AB015846 AB011207 AB015848 Z49096 int β-lactams,Enterobacteriaceae,  99-102³ trimethoprim sul aminoglycosides,Pseudomonads 103-106³ antiseptic, chloramphenicol ermA¹⁴ Macrolides,Staphylococcus sp. 113⁴ lincosamides, streptogramin B ermB¹⁴ Macrolides,Enterobacteriaceae, 114⁴ lincosamides, Staphylococcus sp. streptograminB Enterococcus sp. Streptococcus sp. ermC¹⁴ Macrolides,Enterobacteriaceae, 115⁴ lincosamides, Staphylococcus sp. streptograminB ereA¹² Macrolides Enterobacteriaceae, M11277 Staphylococcus sp. E01199AF099140 ereB¹² Macrolides Enterobacteriaceae A15097 Staphylococcus sp.X03988 msrA¹² Macrolides Staphylococcus sp. 77-80³ mefA, mefE⁸Macrolides Streptococcus sp. U70055 U83667 mphA⁸ MacrolidesEnterobacteriaceae, D16251 Staphylococcus sp. U34344 U36578 linA/linA′⁹Lincosamides Staphylococcus sp. J03947 M14039 A15070 E01245 linB¹⁰Lincosamides Enterococcus faecium AF110130 AJ238249 vga¹⁵ StreptrograminStaphylococcus sp. M90056 89-90³ U82085 vgb¹⁵ StreptrograminStaphylococcus sp. M36022 M20219 AF015628 vat¹⁵ StreptrograminStaphylococcus sp. L07778 87-88³ vatB¹⁵ Streptrogramin Staphylococcussp. U19459 L38809 satA¹⁵ Streptrogramin Enterococcus faecium L1203381-82³ mupA¹² Mupirocin Staphylococcus aureus X75439 X59478 X59477gyrA¹⁶ Quinolones Gram-positive and X95718 1255, 1607-1608,gram-negative bacteria X06744 1764-1776, X57174 2013-2014, X168172277-2280 X71437 AF065152 AF060881 D32252 parC/grlA¹⁶ QuinolonesGram-positive and AB005036 1777-1785  gram-negative bacteria AF056287X95717 AF129764 AB017811 AF065152 parE/grlB¹⁶ Quinolones Gram-positivebacteria X95717 AF065153 AF058920 norA¹⁶ Quinolones Staphylococcus sp.D90119 M80252 M97169 mexR (nalB)¹⁶ Quinolones Pseudomonas aeruginosaU23763 nfxB¹⁶ Quinolones Pseudomonas aeruginosa X65646 cat¹²Chloramphenicol Gram-positive and M55620 gram-negative bacteria X15100A24651 M28717 A00568 A00569 X74948 Y00723 A24362 A00569 M93113 M62822M58516 V01277 X02166 M77169 X53796 J01841 X07848 ppflo-likeChloramphenicol AF071555 embB¹⁷ Ethambutol Mycobacterium tuberculosisU68480 pncA¹⁷ Pyrazinamide Mycobacterium tuberculosis U59967 rpoB¹⁷Rifampin Mycobacterium tuberculosis AF055891 AF055892 S71246 L27989AF055893 inhA¹⁷ Isoniazid Mycobacterium tuberculosis AF106077 U02492vanA¹² Vancomycin Enterococcus sp. 67-70³ 1049-1057  vanB¹² VancomycinEnterococcus sp. 116⁴ vanC1¹² Vancomycin Enterococcus gallinarum 117⁴1058-1059  vanC2¹² Vancomycin Enterococcus casseliflavus U945211060-1063  U94522 U94523 U94524 U94525 L29638 vanC3¹² VancomycinEnterococcus flavescens L29639 1064-1066  U72706 vanD¹⁸ VancomycinEnterococcus faecium AF130997 vanE¹² Vancomycin Enterococcus faeciumAF136925 tetB¹⁹ Tetracycline Gram-negative bacteria J01830 AF162223AP000342 S83213 U81141 V00611 tetM¹⁹ Tetracycline Gram-negative andX52632 Gram-positive bacteria AF116348 U50983 X92947 M211136 U08812X04388 sul II²⁰ Sulfonamides Gram-negative bacteria M36657 AF017389AF017391 dhfrIa²⁰ Trimethoprim Gram-negative bacteria AJ238350 X17477K00052 U09476 X00926 dhfrIb²⁰ Trimethoprim Gram-negative bacteria Z50805Z50804 dhfrV²⁰ Trimethoprim Gram-negative bacteria X12868 dhfrVI²⁰Trimethoprim Gram-negative bacteria Z86002 dhfrVII²⁰ TrimethoprimGram-negative bacteria U31119 AF139109 X58425 dhfrVIII²⁰ TrimethoprimGram-negative bacteria U10186 U09273 dhfrIX²⁰ Trimethoprim Gram-negativebacteria X57730 dhfrXII²⁰ Trimethoprim Gram-negative bacteria Z21672AF175203 AF180731 M84522 dhfrXIII²⁰ Trimethoprim Gram-negative bacteriaZ50802 dhfrXV²⁰ Trimethoprim Gram-negative bacteria Z83331 dhfrXVII²⁰Trimethoprim Gram-negative bacteria AF170088 AF180469 AF169041 dfrA²⁰Trimethoprim Staphylococcus sp. AF045472 U40259 AF051916 X13290 Y07536Z16422 Z48233 ¹Bacteria having high incidence for the specifiedantibiotic resistance gene. The presence of the antibiotic resistancegenes in other bacteria is not excluded. ²Shaw, K. J., P. N. Rather, R.S. Hare, and G. H. Miller. 1993. Molecular genetics of aminoglycosideresistance genes and familial relationships of theaminoglycoside-modifying enzymes. Microbiol. Rev. 57: 138-163.³Antibiotic resistance genes from our assigned U.S. Pat. No. 6,001,564for which we have selected PCR primer pairs. ⁴These SEQ ID NOs. refer toa previous patent (publication WO98/20157). ⁵Bush, K., G. A. Jacoby andA. Medeiros. 1995. A functional classification scheme for β-lactamaseand its correlation with molecular structure. Antimicrob. Agents.Chemother. 39: 1211-1233. ⁶Nucleotide mutations in bla_(SHV), bla_(TEM),and bla_(OXA), are associated with extended-spectrum β-lactamase orinhibitor-resistant β-lactamase. ⁷Bauerfeind, A., Y. Chong, and K. Lee.1998. Plasmid-encoded AmpC beta-lactamases: how far have we gone 10 earsafter discovery? Yonsei Med. J. 39: 520-525. ⁸Sutcliffe, J., T. Grebe,A. Tait-Kamradt, and L. Wondrack. 1996. Detection oferythromycin-resistant determinants by PCR. Antimicrob. Agent Chemother.40: 2562-2566. ⁹Leclerc, R., A., Brisson-Noël, J. Duval, and P.Courvalin. 1991. Phenotypic expression and genetic heterogeneity oflincosamide inactivation in Staphylococcus sp. Antimicrob. Agents.Chemother. 31: 1887-1891. ¹⁰Bozdogan, B., L. Berrezouga, M.-S. Kuo, D.A. Yurek, K. A. Farley, B. J. Stockman, and R. Leclercq. 1999. A newgene, linB, conferring resistance to lincosamides by nucleotidylation inEnterococcus faecium HM1025. Antimicrob. Agents. Chemother. 43: 925-929.¹¹Cockerill III, F. R. 1999. Genetic methods for assessing antimicrobialresistance. Antimicrob. Agents. Chemother. 43: 199-212. ¹²Tenover, F.C., T. Popovic, and O Olsvik. 1996. Genetic methods for detectingantibacterial resistance genes. pp. 1368-1378. In Murray, P. R., E. J.Baron, M. A. Pfaller, F. C. Tenover, R. H. Yolken (eds). Manual ofclinical microbiology. 6th ed., ASM Press, Washington, D.C. USA¹³Dowson, C. G., T. J. Tracey, and B. G. Spratt. 1994. Origin andmolecular epidemiology of penicillin-binding-protein-mediated resistanceto β-lactam antibiotics. Trends Molec. Microbiol. 2: 361-366. ¹⁴Jensen,L. B., N. Frimodt-Moller, F. M. Aarestrup. 1999. Presence of erm geneclasses in Gram-positive bacteria of animal and human origin in Denmark.FEMS Microbiol. 170: 151-158. ¹⁵Thal, L. A., and M. J. Zervos. 1999.Occurrence and epidemiology of resistance to virginimycin andstreptrogramins. J. Antimicrob. Chemother. 43: 171-176. ¹⁶Martinez J.L., A. Alonso, J. M. Gomez-Gomez, and F. Baquero. 1998. Quinoloneresistance by mutations in chromosomal gyrase genes. Just the tip of theiceberg? J. Antimicrob. Chemother. 42: 683-688 ¹⁷Cockerill III, F. R.1999. Genetic methods for assessing antimicrobial resistance.Antimicrob. Agents. Chemother. 43: 199-212. ¹⁸Casadewall, B. and P.Courvalin. 1999 Characterization of the vanD glycopeptide resistancegene cluster from Enterococcus faecium BM 4339. J. Bacteriol. 181:3644-3648. ¹⁹Roberts, M. C. 1999. Genetic mobility and distribution oftetracycline resistance determinants. Ciba Found. Symp. 207: 206-222.²⁰Huovinen, P., L. Sundström, G. Swedberg, and O. Sköld. 1995.Trimethoprim and sulfonamide resistance. Antimicrob. Agent Chemother.39: 279-289.

TABLE 6 List of bacterial toxins selected for diagnostic purposes.Organism Toxin Accession number Actinobacillus actinomycetemcomitansCytolethal distending toxin (cdtA, cdtB, cdtC) AF006830 Leukotoxin(ltxA) M27399 Actinomyces pyogenes Hemolysin (pyolysin) U84782 Aeromonashydrophila Aerolysin (aerA) M16495 Haemolysin (hlyA) U81555 Cytotonicenterotoxin (alt) L77573 Bacillus anthracis Anthrax toxin (cya) M23179Bacillus cereus Enterotoxin (bceT) D17312 AF192766, AF192767 Enterotoxichemolysin BL AJ237785 Non-haemolytic enterotoxins A, B and C (nhe)Y19005 Bacillus mycoides Hemolytic enterotoxin HBL AJ243150 to AJ243153Bacillus pseudomycoides Hemolytic enterotoxin HBL AJ243154 to AJ243156Bacteroides fragilis Enterotoxin (bftP) U67735 Matrixmetalloprotease/enterotoxin (fragilysin) S75941, AF038459Metalloprotease toxin-2 U90931 AF081785 Metalloprotease toxin-3 AF056297Bordetella bronchiseptica Adenylate cyclase hemolysin (cyaA) Z37112,U22953 Dermonecrotic toxin (dnt) U59687 AB020025 Bordetella pertussisPertussis toxin (S1 subunit, tox) AJ006151 AJ006153 AJ006155 AJ006157AJ006159 AJ007363 M14378, M16494 AJ007364 M13223 X16347 Adenyl cyclase(cya) 18323 Dermonecrotic toxin (dnt) U10527 Campylobacter jejuniCytolethal distending toxin (cdtA, cdtB, cdtC) U51121 Citrobacterfreundii Shiga-like toxin (slt-IIcA) X67514, S53206 Clostridiumbotulinum Botulism toxin (BoNT) (A, B, E and F serotypes X52066, X52088are neurotoxic for humans; the other serotypes X73423 have not beenconsidered) M30196 X70814 X70819 X71343 Z11934 X70817 M81186 X70818X70815 X62089 X62683 S76749 X81714 X70816 X70820 X70281 L35496 M92906Clostridium difficile A toxin (enterotoxin) (tcdA) (cdtA) AB012304AF053400 Y12616 X51797 X17194 M30307 B toxin (cytotoxin) (toxB) (cdtB)Z23277 X53138 Clostridium perfringens Alpha (phospholipase C) (cpa)L43545 L43546 L43547 L43548 X13608 X17300 D10248 Beta (dermonecroticprotein) (cpb) L13198 X83275 L77965 Enterotoxin (cpe) AJ000766 M98037X81849 X71844 Y16009 Enterotoxin pseudogene (not expressed) AF037328AF037329 AF037330 Epsilon toxin (etxD) M80837 M95206 X60694 Iota (Ia andIb) X73562 Lambda (metalloprotease) D45904 Theta (perfringolysin O)M36704 Clostridium sordellii Cytotoxin L X82638 Clostridium tetaniTetanos toxin X06214 X04436 Corynebacterium diphtheriae Diphtheriaetoxin X00703 Corynebacterium pseudotuberculosis Phospholipase C A21336Eikenella corrodens lysine decarboxylase (cadA) U89166 Enterobactercloacae Shiga-like toxin II Z50754, U33502 Enterococcus faecalisCytolysin B (cylB) M38052 Escherichia coli (EHEC) Hemolysin toxin (hlyAand ehxA) AF043471 X94129 X79839 X86087 AB011549 AF074613

TABLE 7 Origin of the nucleic acids and/or sequences in the sequencelisting. SEQ ID NO. Archaeal, bacterial, fungal or parasitical speciesSource Gene* 1 Acinetobacter baumannii This patent tuf 2 Actinomycesmeyeri This patent tuf 3 Aerococcus viridans This patent tuf 4Achromobacter xylosoxidans subsp. denitrificans This patent tuf 5Anaerorhabdus furcosus This patent tuf 6 Bacillus anthracis This patenttuf 7 Bacillus cereus This patent tuf 8 Bacteroides distasonis Thispatent tuf 9 Enterococcus casseliflavus This patent tuf 10Staphylococcus saprophyticus This patent tuf 11 Bacteroides ovatus Thispatent tuf 12 Bartonella henselae This patent tuf 13 Bifidobacteriumadolescentis This patent tuf 14 Bifidobacterium dentium This patent tuf15 Brucella abortus This patent tuf 16 Burkholderia cepacia This patenttuf 17 Cedecea davisae This patent tuf 18 Cedecea neteri This patent tuf19 Cedecea lapagei This patent tuf 20 Chlamydia pneumoniae This patenttuf 21 Chlamydia psittaci This patent tuf 22 Chlamydia trachomatis Thispatent tuf 23 Chryseobacterium meningosepticum This patent tuf 24Citrobacter amalonaticus This patent tuf 25 Citrobacter braakii Thispatent tuf 26 Citrobacter koseri This patent tuf 27 Citrobacter farmeriThis patent tuf 28 Citrobacter freundii This patent tuf 29 Citrobactersedlakii This patent tuf 30 Citrobacter werkmanii This patent tuf 31Citrobacter youngae This patent tuf 32 Clostridium perfringens Thispatent tuf 33 Comamonas acidovorans This patent tuf 34 Corynebacteriumbovis This patent tuf 35 Corynebacterium cervicis This patent tuf 36Corynebacterium flavescens This patent tuf 37 Corynebacterium kutscheriThis patent tuf 38 Corynebacterium minutissimum This patent tuf 39Corynebacterium mycetoides This patent tuf 40 Corynebacteriumpseudogenitalium This patent tuf 41 Corynebacterium renale This patenttuf 42 Corynebacterium ulcerans This patent tuf 43 Corynebacteriumurealyticum This patent tuf 44 Corynebacterium xerosis This patent tuf45 Coxiella burnetii This patent tuf 46 Edwardsiella hoshinae Thispatent tuf 47 Edwardsiella tarda This patent tuf 48 Eikenella corrodensThis patent tuf 49 Enterobacter aerogenes This patent tuf 50Enterobacter agglomerans This patent tuf 51 Enterobacter amnigenus Thispatent tuf 52 Enterobacter asburiae This patent tuf 53 Enterobactercancerogenus This patent tuf 54 Enterobacter cloacae This patent tuf 55Enterobacter gergoviae This patent tuf 56 Enterobacter hormaechei Thispatent tuf 57 Enterobacter sakazakii This patent tuf 58 Enterococcuscasseliflavus This patent tuf 59 Enterococcus cecorum This patent tuf 60Enterococcus dispar This patent tuf 61 Enterococcus durans This patenttuf 62 Enterococcus faecalis This patent tuf 63 Enterococcus faecalisThis patent tuf 64 Enterococcus faecium This patent tuf 65 Enterococcusflavescens This patent tuf 66 Enterococcus gallinarum This patent tuf 67Enterococcus hirae This patent tuf 68 Enterococcus mundtii This patenttuf 69 Enterococcus pseudoavium This patent tuf 70 Enterococcusraffinosus This patent tuf 71 Enterococcus saccharolyticus This patenttuf 72 Enterococcus solitarius This patent tuf 73 Enterococcuscasseliflavus This patent tuf (C) 74 Staphylococcus saprophyticus Thispatent unknown 75 Enterococcus flavescens This patent tuf (C) 76Enterococcus gallinarum This patent tuf (C) 77 Ehrlichia canis Thispatent tuf 78 Escherichia coli This patent tuf 79 Escherichia fergusoniiThis patent tuf 80 Escherichia hermannii This patent tuf 81 Escherichiavulneris This patent tuf 82 Eubacterium lentum This patent tuf 83Eubacterium nodatum This patent tuf 84 Ewingella americana This patenttuf 85 Francisella tularensis This patent tuf 86 Fusobacterium nucleatumsubsp. polymorphum This patent tuf 87 Gemella haemolysans This patenttuf 88 Gemella morbillorum This patent tuf 89 Haemophilusactinomycetemcomitans This patent tuf 90 Haemophilus aphrophilus Thispatent tuf 91 Haemophilus ducreyi This patent tuf 92 Haemophilushaemolyticus This patent tuf 93 Haemophilus parahaemolyticus This patenttuf 94 Haemophilus parainfluenzae This patent tuf 95 Haemophilusparaphrophilus This patent tuf 96 Haemophilus segnis This patent tuf 97Hafnia alvei This patent tuf 98 Kingella kingae This patent tuf 99Klebsiella omithinolytica This patent tuf 100 Klebsiella oxytoca Thispatent tuf 101 Klebsiella planticola This patent tuf 102 Klebsiellapneumoniae subsp. ozaenae This patent tuf 103 Klebsiella pneumoniaepneumoniae This patent tuf 104 Klebsiella pneumoniae subsp.rhinoscleromatis This patent tuf 105 Kluyvera ascorbata This patent tuf106 Kluyvera cryocrescens This patent tuf 107 Kluyvera georgiana Thispatent tuf 108 Lactobacillus casei subsp. casei This patent tuf 109Lactococcus lactis subsp. lactis This patent tuf 110 Leclerciaadecarboxylata This patent tuf 111 Legionella micdadei This patent tuf112 Legionella pneumophila subsp. pneumophila This patent tuf 113Leminorella grimontii This patent tuf 114 Leminorella richardii Thispatent tuf 115 Leptospira interrogans This patent tuf 116 Megamonashypermegale This patent tuf 117 Mitsuokella multacidus This patent tuf118 Mobiluncus curtisii subsp. holmesii This patent tuf 119 Moellerellawisconsensis This patent tuf 120 Moraxella catarrhalis This patent tuf121 Morganella morganii subsp. morganii This patent tuf 122Mycobacterium tuberculosis This patent tuf 123 Neisseria cinerea Thispatent tuf 124 Neisseria elongata subsp. elongata This patent tuf 125Neisseria flavescens This patent tuf 126 Neisseria gonorrhoeae Thispatent tuf 127 Neisseria lactamica This patent tuf 128 Neisseriameningitidis This patent tuf 129 Neisseria mucosa This patent tuf 130Neisseria sicca This patent tuf 131 Neisseria subflava This patent tuf132 Neisseria weaveri This patent tuf 133 Ochrobactrum anthropi Thispatent tuf 134 Pantoea agglomerans This patent tuf 135 Pantoea dispersaThis patent tuf 136 Pasteurella multocida This patent tuf 137Peptostreptococcus anaerobius This patent tuf 138 Peptostreptococcusasaccharolyticus This patent tuf 139 Peptostreptococcus prevotii Thispatent tuf 140 Porphyromonas asaccharolytica This patent tuf 141Porphyromonas gingivalis This patent tuf 142 Pragia fontium This patenttuf 143 Prevotella melaninogenica This patent tuf 144 Prevotella oralisThis patent tuf 145 Propionibacterium acnes This patent tuf 146 Proteusmirabilis This patent tuf 147 Proteus penneri This patent tuf 148Proteus vulgaris This patent tuf 149 Providencia alcalifaciens Thispatent tuf 150 Providencia rettgeri This patent tuf 151 Providenciarustigianii This patent tuf 152 Providencia stuartii This patent tuf 153Pseudomonas aeruginosa This patent tuf 154 Pseudomonas fluorescens Thispatent tuf 155 Pseudomonas stutzeri This patent tuf 156 Psychrobacterphenylpyruvicum This patent tuf 157 Rahnella aquatilis This patent tuf158 Salmonella choleraesuis subsp. arizonae This patent tuf 159Salmonella choleraesuis subsp. choleraesuis This patent tuf serotypeCholeraesuis 160 Salmonella choleraesuis subsp. diarizonae This patenttuf 161 Salmonella choleraesuis subsp. choleraesuis This patent tufserotype Heidelberg 162 Salmonella choleraesuis subsp. houtenae Thispatent tuf 163 Salmonella choleraesuis subsp. indica This patent tuf 164Salmonella choleraesuis subsp. salamae This patent tuf 165 Salmonellacholeraesuis subsp. choleraesuis This patent tuf serotype Typhi 166Serratia fonticola This patent tuf 167 Serratia liquefaciens This patenttuf 168 Serratia marcescens This patent tuf 169 Serratia odorifera Thispatent tuf 170 Serratia plymuthica This patent tuf 171 Serratia rubidaeaThis patent tuf 172 Shigella boydii This patent tuf 173 Shigelladysenteriae This patent tuf 174 Shigella flexneri This patent tuf 175Shigella sonnei This patent tuf 176 Staphylococcus aureus This patenttuf 177 Staphylococcus aureus This patent tuf 178 Staphylococcus aureusThis patent tuf 179 Staphylococcus aureus This patent tuf 180Staphylococcus aureus subsp. aureus This patent tuf 181 Staphylococcusauricularis This patent tuf 182 Staphylococcus capitis subsp. capitisThis patent tuf 183 Macrococcus caseolyticus This patent tuf 184Staphylococcus cohnii subsp. cohnii This patent tuf 185 Staphylococcusepidermidis This patent tuf 186 Staphylococcus haemolyticus This patenttuf 187 Staphylococcus warneri This patent tuf 188 Staphylococcushaemolyticus This patent tuf 189 Staphylococcus haemolyticus This patenttuf 190 Staphylococcus haemolyticus This patent tuf 191 Staphylococcushominis subsp. hominis This patent tuf 192 Staphylococcus warneri Thispatent tuf 193 Staphylococcus hominis This patent tuf 194 Staphylococcushominis This patent tuf 195 Staphylococcus hominis This patent tuf 196Staphylococcus hominis This patent tuf 197 Staphylococcus lugdunensisThis patent tuf 198 Staphylococcus saprophyticus This patent tuf 199Staphylococcus saprophyticus This patent tuf 200 Staphylococcussaprophyticus This patent tuf 201 Staphylococcus sciuri subsp. sciuriThis patent tuf 202 Staphylococcus warneri This patent tuf 203Staphylococcus warneri This patent tuf 204 Bifidobacterium longum Thispatent tuf 205 Stenotrophomonas maltophilia This patent tuf 206Streptococcus acidominimus This patent tuf 207 Streptococcus agalactiaeThis patent tuf 208 Streptococcus agalactiae This patent tuf 209Streptococcus agalactiae This patent tuf 210 Streptococcus agalactiaeThis patent tuf 211 Streptococcus anginosus This patent tuf 212Streptococcus bovis This patent tuf 213 Streptococcus anginosus Thispatent tuf 214 Streptococcus cricetus This patent tuf 215 Streptococcuscristatus This patent tuf 216 Streptococcus downei This patent tuf 217Streptococcus dysgalactiae This patent tuf 218 Streptococcus equi subsp.equi This patent tuf 219 Streptococcus ferus This patent tuf 220Streptococcus gordonii This patent tuf 221 Streptococcus anginosus Thispatent tuf 222 Streptococcus macacae This patent tuf 223 Streptococcusgordonii This patent tuf 224 Streptococcus mutans This patent tuf 225Streptococcus parasanguinis This patent tuf 226 Streptococcus ratti Thispatent tuf 227 Streptococcus sanguinis This patent tuf 228 Streptococcussobrinus This patent tuf 229 Streptococcus suis This patent tuf 230Streptococcus uberis This patent tuf 231 Streptococcus vestibularis Thispatent tuf 232 Tatumella ptyseos This patent tuf 233 Trabulsiellaguamensis This patent tuf 234 Veillonella parvula This patent tuf 235Yersinia enterocolitica This patent tuf 236 Yersinia frederiksenii Thispatent tuf 237 Yersinia intermedia This patent tuf 238 Yersinia pestisThis patent tuf 239 Yersinia pseudotuberculosis This patent tuf 240Yersinia rohdei This patent tuf 241 Yokenella regensburgei This patenttuf 242 Achromobacter xylosoxidans subsp. denitrificans This patent atpD243 Acinetobacter baumannii This patent atpD 244 Acinetobacter lwoffiiThis patent atpD 245 Staphylococcus saprophyticus This patent atpD 246Alcaligenes faecalis subsp. faecalis This patent atpD 247 Bacillusanthracis This patent atpD 248 Bacillus cereus This patent atpD 249Bacteroides distasonis This patent atpD 250 Bacteroides ovatus Thispatent atpD 251 Leclercia adecarboxylata This patent atpD 252Stenotrophomonas maltophilia This patent atpD 253 Bartonella henselaeThis patent atpD 254 Bifidobacterium adolescentis This patent atpD 255Brucella abortus This patent atpD 256 Cedecea davisae This patent atpD257 Cedecea lapagei This patent atpD 258 Cedecea neteri This patent atpD259 Chryseobacterium meningosepticum This patent atpD 260 Citrobacteramalonaticus This patent atpD 261 Citrobacter braakii This patent atpD262 Citrobacter koseri This patent atpD 263 Citrobacter farmeri Thispatent atpD 264 Citrobacter freundii This patent atpD 265 Citrobacterkoseri This patent atpD 266 Citrobacter sedlakii This patent atpD 267Citrobacter werkmanii This patent atpD 268 Citrobacter youngae Thispatent atpD 269 Clostridium innocuum This patent atpD 270 Clostridiumperfringens This patent atpD 272 Corynebacterium diphtheriae This patentatpD 273 Corynebacterium pseudodiphtheriticum This patent atpD 274Corynebacterium ulcerans This patent atpD 275 Corynebacteriumurealyticum This patent atpD 276 Coxiella burnetii This patent atpD 277Edwardsiella hoshinae This patent atpD 278 Edwardsiella tarda Thispatent atpD 279 Eikenella corrodens This patent atpD 280 Enterobacteragglomerans This patent atpD 281 Enterobacter amnigenus This patent atpD282 Enterobacter asburiae This patent atpD 283 Enterobacter cancerogenusThis patent atpD 284 Enterobacter cloacae This patent atpD 285Enterobacter gergoviae This patent atpD 286 Enterobacter hormaechei Thispatent atpD 287 Enterobacter sakazakii This patent atpD 288 Enterococcusavium This patent atpD 289 Enterococcus casseliflavus This patent atpD290 Enterococcus durans This patent atpD 291 Enterococcus faecalis Thispatent atpD 292 Enterococcus faecium This patent atpD 293 Enterococcusgallinarum This patent atpD 294 Enterococcus saccharolyticus This patentatpD 295 Escherichia fergusonii This patent atpD 296 Escherichiahermannii This patent atpD 297 Escherichia vulneris This patent atpD 298Eubacterium lentum This patent atpD 299 Ewingella americana This patentatpD 300 Francisella tularensis This patent atpD 301 Fusobacteriumgonidiaformans This patent atpD 302 Fusobacterium necrophorum subsp.necrophorum This patent atpD 303 Fusobacterium nucleatum subsp.polymorphum This patent atpD 304 Gardnerella vaginalis This patent atpD305 Gemella haemolysans This patent atpD 306 Gemella morbillorum Thispatent atpD 307 Haemophilus ducreyi This patent atpD 308 Haemophilushaemolyticus This patent atpD 309 Haemophilus parahaemolyticus Thispatent atpD 310 Haemophilus parainfluenzae This patent atpD 311 Hafniaalvei This patent atpD 312 Kingella kingae This patent atpD 313Klebsiella pneumoniae subsp. ozaenae This patent atpD 314 Klebsiellaornithinolytica This patent atpD 315 Klebsiella oxytoca This patent atpD316 Klebsiella planticola This patent atpD 317 Klebsiella pneumoniaesubsp. pneumoniae This patent atpD 318 Kluyvera ascorbata This patentatpD 319 Kluyvera cryocrescens This patent atpD 320 Kluyvera georgianaThis patent atpD 321 Lactobacillus acidophilus This patent atpD 322Legionella pneumophila subsp. pneumophila This patent atpD 323Leminorella grimontii This patent atpD 324 Listeria monocytogenes Thispatent atpD 325 Micrococcus lylae This patent atpD 326 Moellerellawisconsensis This patent atpD 327 Moraxella catarrhalis This patent atpD328 Moraxella osloensis This patent atpD 329 Morganella morganii subsp.morganii This patent atpD 330 Pantoea agglomerans This patent atpD 331Pantoea dispersa This patent atpD 332 Pasteurella multocida This patentatpD 333 Pragia fontium This patent atpD 334 Proteus mirabilis Thispatent atpD 335 Proteus vulgaris This patent atpD 336 Providenciaalcalifaciens This patent atpD 337 Providencia rettgeri This patent atpD338 Providencia rustigianii This patent atpD 339 Providencia stuartiiThis patent atpD 340 Psychrobacter phenylpyruvicum This patent atpD 341Rahnella aquatilis This patent atpD 342 Salmonella choleraesuis subsp.arizonae This patent atpD 343 Salmonella choleraesuis subsp.choleraesuis This patent atpD serotype Choleraesuis 344 Salmonellacholeraesuis subsp. diarizonae This patent atpD 345 Salmonellacholeraesuis subsp. houtenae This patent atpD 346 Salmonellacholeraesuis subsp. indica This patent atpD 347 Salmonella choleraesuissubsp. choleraesuis This patent atpD serotype Paratyphi A 348 Salmonellacholeraesuis subsp. choleraesuis This patent atpD serotype Paratyphi B349 Salmonella choleraesuis subsp. salamae This patent atpD 350Salmonella choleraesuis subsp. choleraesuis This patent atpD serotypeTyphi 351 Salmonella choleraesuis subsp. choleraesuis This patent atpDserotype Typhimurium 352 Salmonella choleraesuis subsp. choleraesuisThis patent atpD serotype Virchow 353 Serratia ficaria This patent atpD354 Serratia fonticola This patent atpD 355 Serratia grimesii Thispatent atpD 356 Serratia liquefaciens This patent atpD 357 Serratiamarcescens This patent atpD 358 Serratia odorifera This patent atpD 359Serratia plymuthica This patent atpD 360 Serratia rubidaea This patentatpD 361 Pseudomonas putida This patent atpD 362 Shigella boydii Thispatent atpD 363 Shigella dysenteriae This patent atpD 364 Shigellaflexneri This patent atpD 365 Shigella sonnei This patent atpD 366Staphylococcus aureus This patent atpD 367 Staphylococcus auricularisThis patent atpD 368 Staphylococcus capitis subsp. capitis This patentatpD 369 Staphylococcus cohnii subsp. cohnii This patent atpD 370Staphylococcus epidermidis This patent atpD 371 Staphylococcushaemolyticus This patent atpD 372 Staphylococcus hominis subsp. hominisThis patent atpD 373 Staphylococcus hominis This patent atpD 374Staphylococcus lugdunensis This patent atpD 375 Staphylococcussaprophyticus This patent atpD 376 Staphylococcus simulans This patentatpD 377 Staphylococcus warneri This patent atpD 378 Streptococcusacidominimus This patent atpD 379 Streptococcus agalactiae This patentatpD 380 Streptococcus agalactiae This patent atpD 381 Streptococcusagalactiae This patent atpD 382 Streptococcus agalactiae This patentatpD 383 Streptococcus agalactiae This patent atpD 384 Streptococcusdysgalactiae This patent atpD 385 Streptococcus equi subsp. equi Thispatent atpD 386 Streptococcus anginosus This patent atpD 387Streptococcus salivarius This patent atpD 388 Streptococcus suis Thispatent atpD 389 Streptococcus uberis This patent atpD 390 Tatumellaptyseos This patent atpD 391 Trabulsiella guamensis This patent atpD 392Yersinia bercovieri This patent atpD 393 Yersinia enterocolitica Thispatent atpD 394 Yersinia frederiksenii This patent atpD 395 Yersiniaintermedia This patent atpD 396 Yersinia pseudotuberculosis This patentatpD 397 Yersinia rohdei This patent atpD 398 Yokenella regensburgeiThis patent atpD 399 Yarrowia lipolytica This patent tuf (EF-1) 400Absidia corymbifera This patent tuf (EF-1) 401 Alternaria alternata Thispatent tuf (EF-1) 402 Aspergillus flavus This patent tuf (EF-1) 403Aspergillus fumigatus This patent tuf (EF-1) 404 Aspergillus fumigatusThis patent tuf (EF-1) 405 Aspergillus niger This patent tuf (EF-1) 406Blastoschizomyces capitatus This patent tuf (EF-1) 407 Candida albicansThis patent tuf (EF-1) 408 Candida albicans This patent tuf (EF-1) 409Candida albicans This patent tuf (EF-1) 410 Candida albicans This patenttuf (EF-1) 411 Candida albicans This patent tuf (EF-1) 412 Candidadubliniensis This patent tuf (EF-1) 413 Candida catenulata This patenttuf (EF-1) 414 Candida dubliniensis This patent tuf (EF-1) 415 Candidadubliniensis This patent tuf (EF-1) 416 Candida famata This patent tuf(EF-1) 417 Candida glabrata WO98/20157 tuf (EF-1) 418 Candidaguilliermondii This patent tuf (EF-1) 419 Candida haemulonii This patenttuf (EF-1) 420 Candida inconspicua This patent tuf (EF-1) 421 Candidakefyr This patent tuf (EF-1) 422 Candida krusei WO98/20157 tuf (EF-1)423 Candida lambica This patent tuf (EF-1) 424 Candida lusitaniae Thispatent tuf (EF-1) 425 Candida norvegensis This patent tuf (EF-1) 426Candida parapsilosis WO98/20157 tuf (EF-1) 427 Candida rugosa Thispatent tuf (EF-1) 428 Candida sphaerica This patent tuf (EF-1) 429Candida tropicalis WO98/20157 tuf (EF-1) 430 Candida utilis This patenttuf (EF-1) 431 Candida viswanathii This patent tuf (EF-1) 432 Candidazeylanoides This patent tuf (EF-1) 433 Coccidioides immitis This patenttuf (EF-1) 434 Cryptococcus albidus This patent tuf (EF-1) 435 Exophialajeanselmei This patent tuf (EF-1) 436 Fusarium oxysporum This patent tuf(EF-1) 437 Geotrichum sp. This patent tuf (EF-1) 438 Histoplasmacapsulatum This patent tuf (EF-1) 439 Issatchenkia orientalis KudrjanzevThis patent tuf (EF-1) 440 Malassezia furfur This patent tuf (EF-1) 441Malassezia pachydermatis This patent tuf (EF-1) 442 Malbrancheafilamentosa This patent tuf (EF-1) 443 Metschnikowia pulcherrima Thispatent tuf (EF-1) 444 Paecilomyces lilacinus This patent tuf (EF-1) 445Paracoccidioides brasiliensis This patent tuf (EF-1) 446 Penicilliummarneffei This patent tuf (EF-1) 447 Pichia anomala This patent tuf(EF-1) 448 Pichia anomala This patent tuf (EF-1) 449 Pseudallescheriaboydii This patent tuf (EF-1) 450 Rhizopus oryzae This patent tuf (EF-1)451 Rhodotorula minuta This patent tuf (EF-1) 452 Sporobolomycessalmonicolor This patent tuf (EF-1) 453 Sporothrix schenckii This patenttuf (EF-1) 454 Stephanoascus ciferrii This patent tuf (EF-1) 455Trichophyton mentagrophytes This patent tuf (EF-1) 456 Trichosporoncutaneum This patent tuf (EF-1) 457 Wangiella dermatitidis This patenttuf (EF-1) 458 Aspergillus fumigatus This patent atpD 459Blastoschizomyces capitatus This patent atpD 460 Candida albicans Thispatent atpD 461 Candida dubliniensis This patent atpD 462 Candida famataThis patent atpD 463 Candida glabrata This patent atpD 464 Candidaguilliermondii This patent atpD 465 Candida haemulonii This patent atpD466 Candida inconspicua This patent atpD 467 Candida kefyr This patentatpD 468 Candida krusei This patent atpD 469 Candida lambica This patentatpD 470 Candida lusitaniae This patent atpD 471 Candida norvegensisThis patent atpD 472 Candida parapsilosis This patent atpD 473 Candidarugosa This patent atpD 474 Candida sphaerica This patent atpD 475Candida tropicalis This patent atpD 476 Candida utilis This patent atpD477 Candida viswanathii This patent atpD 478 Candida zeylanoides Thispatent atpD 479 Coccidioides immitis This patent atpD 480 Cryptococcusalbidus This patent atpD 481 Fusarium oxysporum This patent atpD 482Geotrichum sp. This patent atpD 483 Histoplasma capsulatum This patentatpD 484 Malassezia furfur This patent atpD 485 Malassezia pachydermatisThis patent atpD 486 Metschnikowia pulcherrima This patent atpD 487Penicillium marneffei This patent atpD 488 Pichia anomala This patentatpD 489 Pichia anomala This patent atpD 490 Rhodotorula minuta Thispatent atpD 491 Rhodotorula mucilaginosa This patent atpD 492Sporobolomyces salmonicolor This patent atpD 493 Sporothrix schenckiiThis patent atpD 494 Stephanoascus ciferrii This patent atpD 495Trichophyton mentagrophytes This patent atpD 496 Wangiella dermatitidisThis patent atpD 497 Yarrowia lipolytica This patent atpD 498Aspergillus fumigatus This patent tuf (M) 499 Blastoschizomycescapitatus This patent tuf (M) 500 Candida rugosa This patent tuf (M) 501Coccidioides immitis This patent tuf (M) 502 Fusarium oxysporum Thispatent tuf (M) 503 Histoplasma capsulatum This patent tuf (M) 504Paracoccidioides brasiliensis This patent tuf (M) 505 Penicilliummarneffei This patent tuf (M) 506 Pichia anomala This patent tuf (M) 507Trichophyton mentagrophytes This patent tuf (M) 508 Yarrowia lipolyticaThis patent tuf (M) 509 Babesia bigemina This patent tuf (EF-1) 510Babesia bovis This patent tuf (EF-1) 511 Crithidia fasciculata Thispatent tuf (EF-1) 512 Entamoeba histolytica This patent tuf (EF-1) 513Giardia lamblia This patent tuf (EF-1) 514 Leishmania tropica Thispatent tuf (EF-1) 515 Leishmania aethiopica This patent tuf (EF-1) 516Leishmania tropica This patent tuf (EF-1) 517 Leishmania donovani Thispatent tuf (EF-1) 518 Leishmania infantum This patent tuf (EF-1) 519Leishmania enriettii This patent tuf (EF-1) 520 Leishmania gerbilli Thispatent tuf (EF-1) 521 Leishmania hertigi This patent tuf (EF-1) 522Leishmania major This patent tuf (EF-1) 523 Leishmania amazonensis Thispatent tuf (EF-1) 524 Leishmania mexicana This patent tuf (EF-1) 525Leishmania tarentolae This patent tuf (EF-1) 526 Leishmania tropica Thispatent tuf (EF-1) 527 Neospora caninum This patent tuf (EF-1) 528Trichomonas vaginalis This patent tuf (EF-1) 529 Trypanosoma bruceisubsp. brucei This patent tuf (EF-1) 530 Crithidia fasciculata Thispatent atpD 531 Leishmania tropica This patent atpD 532 Leishmaniaaethiopica This patent atpD 533 Leishmania donovani This patent atpD 534Leishmania infantum This patent atpD 535 Leishmania gerbilli This patentatpD 536 Leishmania hertigi This patent atpD 537 Leishmania major Thispatent atpD 538 Leishmania amazonensis This patent atpD 607 Enterococcusfaecalis WO98/20157 tuf 608 Enterococcus faecium WO98/20157 tuf 609Enterococcus gallinarum WO98/20157 tuf 610 Haemophilus influenzaeWO98/20157 tuf 611 Staphylococcus epidermidis WO98/20157 tuf 612Salmonella choleraesuis subsp. choleraesuis This patent tuf serotypeParatyphi A 613 Serratia ficaria This patent tuf 614 Enterococcusmalodoratus This patent tuf (C) 615 Enterococcus durans This patent tuf(C) 616 Enterococcus pseudoavium This patent tuf (C) 617 Enterococcusdispar This patent tuf (C) 618 Enterococcus avium This patent tuf (C)619 Saccharomyces cerevisiae Database tuf (M) 621 Enterococcus faeciumThis patent tuf (C) 622 Saccharomyces cerevisiae This patent tuf (EF-1)623 Cryptococcus neoformans This patent tuf (EF-1) 624 Candida albicansWO98/20157 tuf (EF-1) 662 Corynebacterium diphtheriae WO98/20157 tuf 663Candida catenulata This patent atpD 665 Saccharomyces cerevisiaeDatabase tuf (EF-1) 666 Saccharomyces cerevisiae Database atpD 667Trypanosoma cruzi This patent atpD 668 Corynebacterium glutamicumDatabase tuf 669 Escherichia coli Database atpD 670 Helicobacter pyloriDatabase atpD 671 Clostridium acetobutylicum Database atpD 672 Cytophagalytica Database atpD 673 Ehrlichia risticii This patent atpD 674 Vibriocholerae This patent atpD 675 Vibrio cholerae This patent tuf 676Leishmania enriettii This patent atpD 677 Babesia microti This patenttuf (EF-1) 678 Cryptococcus neoformans This patent atpD 679 Cryptococcusneoformans This patent atpD 680 Cunninghamella bertholletiae This patentatpD 684 Candida tropicalis Database atpD (V) 685 Enterococcus hiraeDatabase atpD (V) 686 Chlamydia pneumoniae Database atpD (V) 687Halobacterium salinarum Database atpD (V) 688 Homo sapiens Database atpD(V) 689 Plasmodium falciparum Database atpD (V) 690 Saccharomycescerevisiae Database atpD (V) 691 Schizosaccharomyces pombe Database atpD(V) 692 Trypanosoma congolense Database atpD (V) 693 Thermusthermophilus Database atpD (V) 698 Escherichia coli WO98/20157 tuf 709Borrelia burgdorferi Database atpD (V) 710 Treponema pallidum DatabaseatpD (V) 711 Chlamydia trachomatis Genome project atpD (V) 712Enterococcus faecalis Genome project atpD (V) 713 Methanosarcina barkeriDatabase atpD (V) 714 Methanococcus jannaschii Database atpD (V) 715Porphyromonas gingivalis Genome project atpD (V) 716 Streptococcuspneumoniae Genome project atpD (V) 717 Burkholderia mallei This patenttuf 718 Burkholderia pseudomallei This patent tuf 719 Clostridiumbeijerinckii This patent tuf 720 Clostridium innocuum This patent tuf721 Clostridium novyi This patent tuf 722 Clostridium septicum Thispatent tuf 723 Clostridium tertium This patent tuf 724 Clostridiumtetani This patent tuf 725 Enterococcus malodoratus This patent tuf 726Enterococcus sulfureus This patent tuf 727 Lactococcus garvieae Thispatent tuf 728 Mycoplasma pirum This patent tuf 729 Mycoplasmasalivarium This patent tuf 730 Neisseria polysaccharea This patent tuf731 Salmonella choleraesuis subsp. choleraesuis This patent tuf serotypeEnteritidis 732 Salmonella choleraesuis subsp. choleraesuis This patenttuf serotype Gallinarum 733 Salmonella choleraesuis subsp. choleraesuisThis patent tuf serotype Paratyphi B 734 Salmonella choleraesuis subsp.choleraesuis This patent tuf serotype Virchow 735 Serratia grimesii Thispatent tuf 736 Clostridium difficile This patent tuf 737 Burkholderiapseudomallei This patent atpD 738 Clostridium bifermentans This patentatpD 739 Clostridium beijerinckii This patent atpD 740 Clostridiumdifficile This patent atpD 741 Clostridium ramosum This patent atpD 742Clostridium septicum This patent atpD 743 Clostridium tertium Thispatent atpD 744 Comamonas acidovorans This patent atpD 745 Klebsiellapneumoniae subsp. rhinoscleromatis This patent atpD 746 Neisseria canisThis patent atpD 747 Neisseria cinerea This patent atpD 748 Neisseriacuniculi This patent atpD 749 Neisseria elongata subsp. elongata Thispatent atpD 750 Neisseria flavescens This patent atpD 751 Neisseriagonorrhoeae This patent atpD 752 Neisseria gonorrhoeae This patent atpD753 Neisseria lactamica This patent atpD 754 Neisseria meningitidis Thispatent atpD 755 Neisseria mucosa This patent atpD 756 Neisseria subflavaThis patent atpD 757 Neisseria weaveri This patent atpD 758 Neisseriaanimalis This patent atpD 759 Proteus penneri This patent atpD 760Salmonella choleraesuis subsp. choleraesuis This patent atpD serotypeEnteritidis 761 Yersinia pestis This patent atpD 762 Burkholderia malleiThis patent atpD 763 Clostridium sordellii This patent atpD 764Clostridium novyi This patent atpD 765 Clostridium botulinum This patentatpD 766 Clostridium histolyticum This patent atpD 767Peptostreptococcus prevotii This patent atpD 768 Absidia corymbiferaThis patent atpD 769 Alternaria alternata This patent atpD 770Aspergillus flavus This patent atpD 771 Mucor circinelloides This patentatpD 772 Piedraia hortai This patent atpD 773 Pseudallescheria boydiiThis patent atpD 774 Rhizopus oryzae This patent atpD 775 Scopulariopsiskoningii This patent atpD 776 Trichophyton mentagrophytes This patentatpD 777 Trichophyton tonsurans This patent atpD 778 Trichosporoncutaneum This patent atpD 779 Cladophialophora carrionii This patent tuf(EF-1) 780 Cunninghamella bertholletiae This patent tuf (EF-1) 781Curvularia lunata This patent tuf (EF-1) 782 Fonsecaea pedrosoi Thispatent tuf (EF-1) 783 Microsporum audouinii This patent tuf (EF-1) 784Mucor circinelloides This patent tuf (EF-1) 785 Phialophora verrucosaThis patent tuf (EF-1) 786 Saksenaea vasiformis This patent tuf (EF-1)787 Syncephalastrum racemosum This patent tuf (EF-1) 788 Trichophytontonsurans This patent tuf (EF-1) 789 Trichophyton mentagrophytes Thispatent tuf (EF-1) 790 Bipolaris hawaiiensis This patent tuf (EF-1) 791Aspergillus fumigatus This patent tuf (M) 792 Trichophytonmentagrophytes This patent tuf (M) 827 Clostridium novyi This patentatpD (V) 828 Clostridium difficile This patent atpD (V) 829 Clostridiumsepticum This patent atpD (V) 830 Clostridium botulinum This patent atpD(V) 831 Clostridium perfringens This patent atpD (V) 832 Clostridiumtetani This patent atpD (V) 833 Streptococcus pyogenes Database atpD (V)834 Babesia bovis This patent atpD (V) 835 Cryptosporidium parvum Thispatent atpD (V) 836 Leishmania infantum This patent atpD (V) 837Leishmania major This patent atpD (V) 838 Leishmania tarentolae Thispatent atpD (V) 839 Trypanosoma brucei This patent atpD (V) 840Trypanosoma cruzi This patent tuf (EF-1) 841 Trypanosoma cruzi Thispatent tuf (EF-1) 842 Trypanosoma cruzi This patent tuf (EF-1) 843Babesia bovis This patent tuf (M) 844 Leishmania aethiopica This patenttuf (M) 845 Leishmania amazonensis This patent tuf (M) 846 Leishmaniadonovani This patent tuf (M) 847 Leishmania infantum This patent tuf (M)848 Leishmania enriettii This patent tuf (M) 849 Leishmania gerbilliThis patent tuf (M) 850 Leishmania major This patent tuf (M) 851Leishmania mexicana This patent tuf (M) 852 Leishmania tarentolae Thispatent tuf (M) 853 Trypanosoma cruzi This patent tuf (M) 854 Trypanosomacruzi This patent tuf (M) 855 Trypanosoma cruzi This patent tuf (M) 856Babesia bigemina This patent atpD 857 Babesia bovis This patent atpD 858Babesia microti This patent atpD 859 Leishmania guyanensis This patentatpD 860 Leishmania mexicana This patent atpD 861 Leishmania tropicaThis patent atpD 862 Leishmania tropica This patent atpD 863 Bordetellapertussis Database tuf 864 Trypanosoma brucei brucei Database tuf (EF-1)865 Cryptosporidium parvum This patent tuf (EF-1) 866 Staphylococcussaprophyticus This patent atpD 867 Zoogloea ramigera This patent atpD868 Staphylococcus saprophyticus This patent tuf 869 Enterococcuscasseliflavus This patent tuf 870 Enterococcus casseliflavus This patenttuf 871 Enterococcus flavescens This patent tuf 872 Enterococcusgallinarum This patent tuf 873 Enterococcus gallinarum This patent tuf874 Staphylococcus haemolyticus This patent tuf 875 Staphylococcusepidermidis This patent tuf 876 Staphylococcus epidermidis This patenttuf 877 Staphylococcus epidermidis This patent tuf 878 Staphylococcusepidermidis This patent tuf 879 Enterococcus gallinarum This patent tuf880 Pseudomonas aeruginosa This patent tuf 881 Enterococcuscasseliflavus This patent tuf 882 Enterococcus casseliflavus This patenttuf 883 Enterococcus faecalis This patent tuf 884 Enterococcus faecalisThis patent tuf 885 Enterococcus faecium This patent tuf 886Enterococcus faecium This patent tuf 887 Zoogloea ramigera This patenttuf 888 Enterococcus faecalis This patent tuf 889 Aspergillus fumigatusThis patent atpD 890 Penicillium marneffei This patent atpD 891Paecilomyces lilacinus This patent atpD 892 Penicillium marneffei Thispatent atpD 893 Sporothrix schenckii This patent atpD 894 Malbrancheafilamentosa This patent atpD 895 Paecilomyces lilacinus This patent atpD896 Aspergillus niger This patent atpD 897 Aspergillus fumigatus Thispatent tuf (EF-1) 898 Penicillium marneffei This patent tuf (EF-1) 899Piedraia hortai This patent tuf (EF-1) 900 Paecilomyces lilacinus Thispatent tuf (EF-1) 901 Paracoccidioides brasiliensis This patent tuf(EF-1) 902 Sporothrix schenckii This patent tuf (EF-1) 903 Penicilliummarneffei This patent tuf (EF-1) 904 Curvularia lunata This patent tuf(M) 905 Aspergillus niger This patent tuf (M) 906 Bipolaris hawaiiensisThis patent tuf (M) 907 Aspergillus flavus This patent tuf (M) 908Alternaria alternata This patent tuf (M) 909 Penicillium marneffei Thispatent tuf (M) 910 Penicillium marneffei This patent tuf (M) 918Escherichia coli Database recA 929 Bacteroides fragilis This patent atpD(V) 930 Bacteroides distasonis This patent atpD (V) 931 Porphyromonasasaccharolytica This patent atpD (V) 932 Listeria monocytogenes Thispatent tuf 939 Saccharomyces cerevisiae Database recA (Rad51) 940Saccharomyces cerevisiae Database recA (Dmc1) 941 Cryptococcus humicolusThis patent atpD 942 Escherichia coli This patent atpD 943 Escherichiacoli This patent atpD 944 Escherichia coli This patent atpD 945Escherichia coli This patent atpD 946 Neisseria polysaccharea Thispatent atpD 947 Neisseria sicca This patent atpD 948 Streptococcus mitisThis patent atpD 949 Streptococcus mitis This patent atpD 950Streptococcus mitis This patent atpD 951 Streptococcus oralis Thispatent atpD 952 Streptococcus pneumoniae This patent atpD 953Streptococcus pneumoniae This patent atpD 954 Streptococcus pneumoniaeThis patent atpD 955 Streptococcus pneumoniae This patent atpD 956Babesia microti This patent atpD (V) 957 Entamoeba histolytica Thispatent atpD (V) 958 Fusobacterium nucleatum subsp. polymorphum Thispatent atpD (V) 959 Leishmania aethiopica This patent atpD (V) 960Leishmania tropica This patent atpD (V) 961 Leishmania guyanensis Thispatent atpD (V) 962 Leishmania donovani This patent atpD (V) 963Leishmania hertigi This patent atpD (V) 964 Leishmania mexicana Thispatent atpD (V) 965 Leishmania tropica This patent atpD (V) 966Peptostreptococcus anaerobius This patent atpD (V) 967 Bordetellapertussis This patent tuf 968 Bordetella pertussis This patent tuf 969Enterococcus columbae This patent tuf 970 Enterococcus flavescens Thispatent tuf 971 Streptococcus pneumoniae This patent tuf 972 Escherichiacoli This patent tuf 973 Escherichia coli This patent tuf 974Escherichia coli This patent tuf 975 Escherichia coli This patent tuf976 Mycobacterium avium This patent tuf 977 Streptococcus pneumoniaeThis patent tuf 978 Mycobacterium gordonae This patent tuf 979Streptococcus pneumoniae This patent tuf 980 Mycobacterium tuberculosisThis patent tuf 981 Staphylococcus warneri This patent tuf 982Streptococcus mitis This patent tuf 983 Streptococcus mitis This patenttuf 984 Streptococcus mitis This patent tuf 985 Streptococcus oralisThis patent tuf 986 Streptococcus pneumoniae This patent tuf 987Enterococcus hirae This patent tuf (C) 988 Enterococcus mundtii Thispatent tuf (C) 989 Enterococcus raffinosus This patent tuf (C) 990Bacillus anthracis This patent recA 991 Prevotella melaninogenica Thispatent recA 992 Enterococcus casseliflavus This patent tuf 993Streptococcus pyogenes Database speA 1002 Streptococcus pyogenesWO98/20157 tuf 1003 Bacillus cereus This patent recA 1004 Streptococcuspneumoniae This patent pbp1a 1005 Streptococcus pneumoniae This patentpbp1a 1006 Streptococcus pneumoniae This patent pbp1a 1007 Streptococcuspneumoniae This patent pbp1a 1008 Streptococcus pneumoniae This patentpbp1a 1009 Streptococcus pneumoniae This patent pbp1a 1010 Streptococcuspneumoniae This patent pbp1a 1011 Streptococcus pneumoniae This patentpbp1a 1012 Streptococcus pneumoniae This patent pbp1a 1013 Streptococcuspneumoniae This patent pbp1a 1014 Streptococcus pneumoniae This patentpbp1a 1015 Streptococcus pneumoniae This patent pbp1a 1016 Streptococcuspneumoniae This patent pbp1a 1017 Streptococcus pneumoniae This patentpbp1a 1018 Streptococcus pneumoniae This patent pbp1a 1019 Streptococcuspneumoniae This patent pbp2b 1020 Streptococcus pneumoniae This patentpbp2b 1021 Streptococcus pneumoniae This patent pbp2b 1022 Streptococcuspneumoniae This patent pbp2b 1023 Streptococcus pneumoniae This patentpbp2b 1024 Streptococcus pneumoniae This patent pbp2b 1025 Streptococcuspneumoniae This patent pbp2b 1026 Streptococcus pneumoniae This patentpbp2b 1027 Streptococcus pneumoniae This patent pbp2b 1028 Streptococcuspneumoniae This patent pbp2b 1029 Streptococcus pneumoniae This patentpbp2b 1030 Streptococcus pneumoniae This patent pbp2b 1031 Streptococcuspneumoniae This patent pbp2b 1032 Streptococcus pneumoniae This patentpbp2b 1033 Streptococcus pneumoniae This patent pbp2b 1034 Streptococcuspneumoniae This patent pbp2x 1035 Streptococcus pneumoniae This patentpbp2x 1036 Streptococcus pneumoniae This patent pbp2x 1037 Streptococcuspneumoniae This patent pbp2x 1038 Streptococcus pneumoniae This patentpbp2x 1039 Streptococcus pneumoniae This patent pbp2x 1040 Streptococcuspneumoniae This patent pbp2x 1041 Streptococcus pneumoniae This patentpbp2x 1042 Streptococcus pneumoniae This patent pbp2x 1043 Streptococcuspneumoniae This patent pbp2x 1044 Streptococcus pneumoniae This patentpbp2x 1045 Streptococcus pneumoniae This patent pbp2x 1046 Streptococcuspneumoniae This patent pbp2x 1047 Streptococcus pneumoniae This patentpbp2x 1048 Streptococcus pneumoniae This patent pbp2x 1049 Enterococcusfaecium This patent vanA 1050 Enterococcus gallinarum This patent vanA1051 Enterococcus faecium This patent vanA 1052 Enterococcus faeciumThis patent vanA 1053 Enterococcus faecium This patent vanA 1054Enterococcus faecalis This patent vanA 1055 Enterococcus gallinarum Thispatent vanA 1056 Enterococcus faecium This patent vanA 1057 Enterococcusflavescens This patent vanA 1058 Enterococcus gallinarum This patentvanC1 1059 Enterococcus gallinarum This patent vanC1 1060 Enterococcuscasseliflavus This patent vanC2 1061 Enterococcus casseliflavus Thispatent vanC2 1062 Enterococcus casseliflavus This patent vanC2 1063Enterococcus casseliflavus This patent vanC2 1064 Enterococcusflavescens This patent vanC3 1065 Enterococcus flavescens This patentvanC3 1066 Enterococcus flavescens This patent vanC3 1067 Enterococcusfaecium This patent vanXY 1068 Enterococcus faecium This patent vanXY1069 Enterococcus faecium This patent vanXY 1070 Enterococcus faecalisThis patent vanXY 1071 Enterococcus gallinarum This patent vanXY 1072Enterococcus faecium This patent vanXY 1073 Enterococcus flavescens Thispatent vanXY 1074 Enterococcus faecium This patent vanXY 1075Enterococcus gallinarum This patent vanXY 1076 Escherichia coli Databasestx₁ 1077 Escherichia coli Database stx₂ 1093 Staphylococcussaprophyticus This patent unknown 1117 Enterococcus faecium DatabasevanB 1138 Enterococcus gallinarum Database vanC1 1139 Enterococcusfaecium Database vanA 1140 Enterococcus casseliflavus Database vanC21141 Enterococcus faecium Database vanHAXY 1169 Streptococcus pneumoniaeDatabase pbp1a 1172 Streptococcus pneumoniae Database pbp2b 1173Streptococcus pneumoniae Database pbp2x 1178 Staphylococcus aureusDatabase mecA 1183 Streptococcus pneumoniae Database hexA 1184Streptococcus pneumoniae This patent hexA 1185 Streptococcus pneumoniaeThis patent hexA 1186 Streptococcus pneumoniae This patent hexA 1187Streptococcus pneumoniae This patent hexA 1188 Streptococcus oralis Thispatent hexA 1189 Streptococcus mitis This patent hexA 1190 Streptococcusmitis This patent hexA 1191 Streptococcus mitis This patent hexA 1198Staphylococcus saprophyticus This patent unknown 1215 Streptococcuspyogenes Database pcp 1230 Escherichia coli Database tuf (EF-G) 1242Enterococcus faecium Database ddl 1243 Enterococcus faecalis DatabasemtlF, mtlD 1244 Staphylococcus aureus subsp. aureus This patent unknown1245 Bacillus anthracis This patent atpD 1246 Bacillus mycoides Thispatent atpD 1247 Bacillus thuringiensis This patent atpD 1248 Bacillusthuringiensis This patent atpD 1249 Bacillus thuringiensis This patentatpD 1250 Bacillus weihenstephanensis This patent atpD 1251 Bacillusthuringiensis This patent atpD 1252 Bacillus thuringiensis This patentatpD 1253 Bacillus cereus This patent atpD 1254 Bacillus cereus Thispatent atpD 1255 Staphylococcus aureus This patent gyrA 1256 Bacillusweihenstephanensis This patent atpD 1257 Bacillus anthracis This patentatpD 1258 Bacillus thuringiensis This patent atpD 1259 Bacillus cereusThis patent atpD 1260 Bacillus cereus This patent atpD 1261 Bacillusthuringiensis This patent atpD 1262 Bacillus thuringiensis This patentatpD 1263 Bacillus thuringiensis This patent atpD 1264 Bacillusthuringiensis This patent atpD 1265 Bacillus anthracis This patent atpD1266 Paracoccidioides brasiliensis This patent tuf (EF-1) 1267Blastomyces dermatitidis This patent tuf (EF-1) 1268 Histoplasmacapsulatum This patent tuf (EF-1) 1269 Trichophyton rubrum This patenttuf (EF-1) 1270 Microsporum canis This patent tuf (EF-1) 1271Aspergillus versicolor This patent tuf (EF-1) 1272 Exophiala moniliaeThis patent tuf (EF-1) 1273 Hortaea werneckii This patent tuf (EF-1)1274 Fusarium solani This patent tuf (EF-1) 1275 Aureobasidium pullulansThis patent tuf (EF-1) 1276 Blastomyces dermatitidis This patent tuf(EF-1) 1277 Exophiala dermatitidis This patent tuf (EF-1) 1278 Fusariummoniliforme This patent tuf (EF-1) 1279 Aspergillus terreus This patenttuf (EF-1) 1280 Aspergillus fumigatus This patent tuf (EF-1) 1281Cryptococcus laurentii This patent tuf (EF-1) 1282 Emmonsia parva Thispatent tuf (EF-1) 1283 Fusarium solani This patent tuf (EF-1) 1284Sporothrix schenckii This patent tuf (EF-1) 1285 Aspergillus nidulansThis patent tuf (EF-1) 1286 Cladophialophora carrionii This patent tuf(EF-1) 1287 Exserohilum rostratum This patent tuf (EF-1) 1288 Bacillusthuringiensis This patent recA 1289 Bacillus thuringiensis This patentrecA 1299 Staphylococcus aureus Database gyrA 1300 Escherichia coliDatabase gyrA 1307 Staphylococcus aureus Database gyrB 1320 Escherichiacoli Database parC (grlA) 1321 Staphylococcus aureus Database parC(grlA) 1328 Staphylococcus aureus Database parE (grlB) 1348 unidentifiedbacterium Database aac2Ia 1351 Pseudomonas aeruginosa Database aac3Ib1356 Serratia marcescens Database aac3IIb 1361 Escherichia coli Databaseaac3IVa 1366 Enterobacter cloacae Database aac3VIa 1371 Citrobacterkoseri Database aac6Ia 1376 Serratia marcescens Database aac6Ic 1381Escherichia coli Database ant3Ia 1386 Staphylococcus aureus Databaseant4Ia 1391 Escherichia coli Database aph3Ia 1396 Escherichia coliDatabase aph3IIa 1401 Enterococcus faecalis Database aph3IIIa 1406Acinetobacter baumannii Database aph3VIa 1411 Pseudomonas aeruginosaDatabase blaCARB 1416 Klebsiella pneumoniae Database blaCMY-2 1423Escherichia coli Database blaCTX-M-1 1428 Salmonella choleraesuis subsp.choleraesuis Database blaCTX-M-2 serotype Typhimurium 1433 Pseudomonasaeruginosa Database blaIMP 1438 Escherichia coli Database blaOXA2 1439Pseudomonas aeruginosa Database blaOXA10 1442 Pseudomonas aeruginosaDatabase blaPER1 1445 Salmonella choleraesuis subsp. choleraesuisDatabase blaPER2 serotype Typhimurium 1452 Staphylococcus epidermidisDatabase dfrA 1461 Escherichia coli Database dhfrIa 1470 Escherichiacoli Database dhfrIb 1475 Escherichia coli Database dhfrV 1480 Proteusmirabilis Database dhfrVI 1489 Escherichia coli Database dhfrVII 1494Escherichia coli Database dhfrVIII 1499 Escherichia coli Database dhfrIX1504 Escherichia coli Database dhfrXII 1507 Escherichia coli DatabasedhfrXIII 1512 Escherichia coli Database dhfrXV 1517 Escherichia coliDatabase dhfrXVII 1518 Acinetobacter lwoffii This patent fusA 1519Acinetobacter lwoffii This patent fusA-tuf spacer 1520 Acinetobacterlwoffii This patent tuf 1521 Haemophilus influenzae This patent fusA1522 Haemophilus influenzae This patent fusA-tuf spacer 1523 Haemophilusinfluenzae This patent tuf 1524 Proteus mirabilis This patent fusA 1525Proteus mirabilis This patent fusA-tuf spacer 1526 Proteus mirabilisThis patent tuf 1527 Campylobacter curvus This patent atpD 1530Escherichia coli Database ereA 1535 Escherichia coli Database ereB 1540Staphylococcus haemolyticus Database linA 1545 Enterococcus faeciumDatabase linB 1548 Streptococcus pyogenes Database mefA 1551Streptococcus pneumoniae Database mefE 1560 Escherichia coli DatabasemphA 1561 Candida albicans This patent tuf (EF-1) 1562 Candidadubliniensis This patent tuf (EF-1) 1563 Candida famata This patent tuf(EF-1) 1564 Candida glabrata This patent tuf (EF-1) 1565 Candidaguilliermondii This patent tuf (EF-1) 1566 Candida haemulonii Thispatent tuf (EF-1) 1567 Candida kefyr This patent tuf (EF-1) 1568 Candidalusitaniae This patent tuf (EF-1) 1569 Candida sphaerica This patent tuf(EF-1) 1570 Candida tropicalis This patent tuf (EF-1) 1571 Candidaviswanathii This patent tuf (EF-1) 1572 Alcaligenes faecalis subsp.faecalis This patent tuf 1573 Prevotella buccalis This patent tuf 1574Succinivibrio dextrinosolvens This patent tuf 1575 Tetragenococcushalophilus This patent tuf 1576 Campylobacter jejuni subsp. jejuni Thispatent atpD 1577 Campylobacter rectus This patent atpD 1578 Enterococcuscasseliflavus This patent fusA 1579 Enterococcus gallinarum This patentfusA 1580 Streptococcus mitis This patent fusA 1585 Enterococcus faeciumDatabase satG 1590 Cloning vector pFW16 Database tetM 1594 Enterococcusfaecium Database vanD 1599 Enterococcus faecalis Database vanE 1600Campylobacter jejuni subsp. doylei This patent atpD 1601 Enterococcussulfureus This patent atpD 1602 Enterococcus solitarius This patent atpD1603 Campylobacter sputorum subsp. sputorum This patent atpD 1604Enterococcus pseudoavium This patent atpD 1607 Klebsiellaornithinolytica This patent gyrA 1608 Klebsiella oxytoca This patentgyrA 1613 Staphylococcus aureus Database vatB 1618 Staphylococcus cohniiDatabase vatC 1623 Staphylococcus aureus Database vga 1628Staphylococcus aureus Database vgaB 1633 Staphylococcus aureus Databasevgb 1638 Aspergillus fumigatus This patent atpD 1639 Aspergillusfumigatus This patent atpD 1640 Bacillus mycoides This patent atpD 1641Bacillus mycoides This patent atpD 1642 Bacillus mycoides This patentatpD 1643 Bacillus pseudomycoides This patent atpD 1644 Bacilluspseudomycoides This patent atpD 1645 Budvicia aquatica This patent atpD1646 Buttiauxella agrestis This patent atpD 1647 Candida norvegica Thispatent atpD 1648 Streptococcus pneumoniae This patent pbp1a 1649Campylobacter lari This patent atpD 1650 Coccidioides immitis Thispatent atpD 1651 Emmonsia parva This patent atpD 1652 Erwinia amylovoraThis patent atpD 1653 Fonsecaea pedrosoi This patent atpD 1654 Fusariummoniliforme This patent atpD 1655 Klebsiella oxytoca This patent atpD1656 Microsporum audouinii This patent atpD 1657 Obesumbacterium proteusThis patent atpD 1658 Paracoccidioides brasiliensis This patent atpD1659 Plesiomonas shigelloides This patent atpD 1660 Shewanellaputrefaciens This patent atpD 1662 Campylobacter curvus This patent tuf1663 Campylobacter rectus This patent tuf 1664 Fonsecaea pedrosoi Thispatent tuf 1666 Microsporum audouinii This patent tuf 1667 Piedraiahortai This patent tuf 1668 Escherichia coli Database tuf 1669 Saksenaeavasiformis This patent tuf 1670 Trichophyton tonsurans This patent tuf1671 Enterobacter aerogenes This patent atpD 1672 Bordetella pertussisDatabase atpD 1673 Arcanobacterium haemolyticum This patent tuf 1674Butyrivibrio fibrisolvens This patent tuf 1675 Campylobacter jejunisubsp. doylei This patent tuf 1676 Campylobacter lari This patent tuf1677 Campylobacter sputorum subsp. sputorum This patent tuf 1678Campylobacter upsaliensis This patent tuf 1679 Globicatella sanguis Thispatent tuf 1680 Lactobacillus acidophilus This patent tuf 1681Leuconostoc mesenteroides subsp. dextranicum This patent tuf 1682Prevotella buccalis This patent tuf 1683 Ruminococcus bromii This patenttuf 1684 Paracoccidioides brasiliensis This patent atpD 1685 Candidanorvegica This patent tuf (EF-1) 1686 Aspergillus nidulans This patenttuf 1687 Aspergillus terreus This patent tuf 1688 Candida norvegica Thispatent tuf 1689 Candida parapsilosis This patent tuf 1702 Streptococcusgordonii WO98/20157 recA 1703 Streptococcus mutans WO98/20157 recA 1704Streptococcus pneumoniae WO98/20157 recA 1705 Streptococcus pyogenesWO98/20157 recA 1706 Streptococcus salivarius subsp. thermophilusWO98/20157 recA 1707 Escherichia coli WO98/20157 oxa 1708 Enterococcusfaecalis WO98/20157 blaZ 1709 Pseudomonas aeruginosa WO98/20157aac6′-IIa 1710 Staphylococcus aureus WO98/20157 ermA 1711 Escherichiacoli WO98/20157 ermB 1712 Staphylococcus aureus WO98/20157 ermC 1713Enterococcus faecalis WO98/20157 vanB 1714 Campylobacter jejuni subsp.jejuni This patent recA 1715 Abiotrophia adiacens WO98/20157 tuf 1716Abiotrophia defectiva WO98/20157 tuf 1717 Corynebacterium accolensWO98/20157 tuf 1718 Corynebacterium genitalium WO98/20157 tuf 1719Corynebacterium jeikeium WO98/20157 tuf 1720 Corynebacteriumpseudodiphtheriticum WO98/20157 tuf 1721 Corynebacterium striatumWO98/20157 tuf 1722 Enterococcus avium WO98/20157 tuf 1723 Gardnerellavaginalis WO98/20157 tuf 1724 Listeria innocua WO98/20157 tuf 1725Listeria ivanovii WO98/20157 tuf 1726 Listeria monocytogenes WO98/20157tuf 1727 Listeria seeligeri WO98/20157 tuf 1728 Staphylococcus aureusWO98/20157 tuf 1729 Staphylococcus saprophyticus WO98/20157 tuf 1730Staphylococcus simulans WO98/20157 tuf 1731 Streptococcus agalactiaeWO98/20157 tuf 1732 Streptococcus pneumoniae WO98/20157 tuf 1733Streptococcus salivarius WO98/20157 tuf 1734 Agrobacterium radiobacterWO98/20157 tuf 1735 Bacillus subtilis WO98/20157 tuf 1736 Bacteroidesfragilis WO98/20157 tuf 1737 Borrelia burgdorferi WO98/20157 tuf 1738Brevibacterium linens WO98/20157 tuf 1739 Chlamydia trachomatisWO98/20157 tuf 1740 Fibrobacter succinogenes WO98/20157 tuf 1741Flavobacterium ferrugineum WO98/20157 tuf 1742 Helicobacter pyloriWO98/20157 tuf 1743 Micrococcus luteus WO98/20157 tuf 1744 Mycobacteriumtuberculosis WO98/20157 tuf 1745 Mycoplasma genitalium WO98/20157 tuf1746 Neisseria gonorrhoeae WO98/20157 tuf 1747 Rickettsia prowazekiiWO98/20157 tuf 1748 Salmonella choleraesuis subsp. choleraesuisWO98/20157 tuf serotype Typhimurium 1749 Shewanella putrefaciensWO98/20157 tuf 1750 Stigmatella aurantiaca WO98/20157 tuf 1751 Thiomonascuprina WO98/20157 tuf 1752 Treponema pallidum WO98/20157 tuf 1753Ureaplasma urealyticum WO98/20157 tuf 1754 Wolinella succinogenesWO98/20157 tuf 1755 Burkholderia cepacia WO98/20157 tuf 1756 Bacillusanthracis This patent recA 1757 Bacillus anthracis This patent recA 1758Bacillus cereus This patent recA 1759 Bacillus cereus This patent recA1760 Bacillus mycoides This patent recA 1761 Bacillus pseudomycoidesThis patent recA 1762 Bacillus thuringiensis This patent recA 1763Bacillus thuringiensis This patent recA 1764 Klebsiella oxytoca Thispatent gyrA 1765 Klebsiella pneumoniae subsp. ozaenae This patent gyrA1766 Klebsiella planticola This patent gyrA 1767 Klebsiella pneumoniaeThis patent gyrA 1768 Klebsiella pneumoniae subsp. pneumoniae Thispatent gyrA 1769 Klebsiella pneumoniae subsp. pneumoniae This patentgyrA 1770 Klebsiella pneumoniae subsp. rhinoscleromatis This patent gyrA1771 Klebsiella terrigena This patent gyrA 1772 Legionella pneumophilasubsp. pneumophila This patent gyrA 1773 Proteus mirabilis This patentgyrA 1774 Providencia rettgeri This patent gyrA 1775 Proteus vulgarisThis patent gyrA 1776 Yersinia enterocolitica This patent gyrA 1777Klebsiella oxytoca This patent parC (grlA) 1778 Klebsiella oxytoca Thispatent parC (grlA) 1779 Klebsiella pneumoniae subsp. ozaenae This patentparC (grlA) 1780 Klebsiella planticola This patent parC (grlA) 1781Klebsiella pneumoniae This patent parC (grlA) 1782 Klebsiella pneumoniaesubsp. pneumoniae This patent parC (grlA) 1783 Klebsiella pneumoniaesubsp. pneumoniae This patent parC (grlA) 1784 Klebsiella pneumoniaesubsp. rhinoscleromatis This patent parC (grlA) 1785 Klebsiellaterrigena This patent parC (grlA) 1786 Bacillus cereus This patent fusA1787 Bacillus cereus This patent fusA 1788 Bacillus anthracis Thispatent fusA 1789 Bacillus cereus This patent fusA 1790 Bacillusanthracis This patent fusA 1791 Bacillus pseudomycoides This patent fusA1792 Bacillus cereus This patent fusA 1793 Bacillus anthracis Thispatent fusA 1794 Bacillus cereus This patent fusA 1795 Bacillusweihenstephanensis This patent fusA 1796 Bacillus mycoides This patentfusA 1797 Bacillus thuringiensis This patent fusA 1798 Bacillusweihenstephanensis This patent fusA-tuf spacer 1799 Bacillusthuringiensis This patent fusA-tuf spacer 1800 Bacillus anthracis Thispatent fusA-tuf spacer 1801 Bacillus pseudomycoides This patent fusA-tufspacer 1802 Bacillus anthracis This patent fusA-tuf spacer 1803 Bacilluscereus This patent fusA-tuf spacer 1804 Bacillus cereus This patentfusA-tuf spacer 1805 Bacillus mycoides This patent fusA-tuf spacer 1806Bacillus cereus This patent fusA-tuf spacer 1807 Bacillus cereus Thispatent fusA-tuf spacer 1808 Bacillus cereus This patent fusA-tuf spacer1809 Bacillus anthracis This patent fusA-tuf spacer 1810 Bacillusmycoides This patent tuf 1811 Bacillus thuringiensis This patent tuf1812 Bacillus cereus This patent tuf 1813 Bacillus weihenstephanensisThis patent tuf 1814 Bacillus anthracis This patent tuf 1815 Bacilluscereus This patent tuf 1816 Bacillus cereus This patent tuf 1817Bacillus anthracis This patent tuf 1818 Bacillus cereus This patent tuf1819 Bacillus anthracis This patent tuf 1820 Bacillus pseudomycoidesThis patent tuf 1821 Bacillus cereus This patent tuf 1822 Streptococcusoralis This patent fusA 1823 Budvicia aquatica This patent fusA 1824Buttiauxella agrestis This patent fusA 1825 Klebsiella oxytoca Thispatent fusA 1826 Plesiomonas shigelloides This patent fusA 1827Shewanella putrefaciens This patent fusA 1828 Obesumbacterium proteusThis patent fusA 1829 Klebsiella oxytoca This patent fusA-tuf spacer1830 Budvicia aquatica This patent fusA-tuf spacer 1831 Plesiomonasshigelloides This patent fusA-tuf spacer 1832 Obesumbacterium proteusThis patent fusA-tuf spacer 1833 Shewanella putrefaciens This patentfusA-tuf spacer 1834 Buttiauxella agrestis This patent fusA-tuf spacer1835 Campylobacter coli This patent tuf 1836 Campylobacter fetus subsp.fetus This patent tuf 1837 Campylobacter fetus subsp. venerealis Thispatent tuf 1838 Buttiauxella agrestis This patent tuf 1839 Klebsiellaoxytoca This patent tuf 1840 Plesiomonas shigelloides This patent tuf1841 Shewanella putrefaciens This patent tuf 1842 Obesumbacteriumproteus This patent tuf 1843 Budvicia aquatica This patent tuf 1844Abiotrophia adiacens This patent atpD 1845 Arcanobacterium haemolyticumThis patent atpD 1846 Basidiobolus ranarum This patent atpD 1847Blastomyces dermatitidis This patent atpD 1848 Blastomyces dermatitidisThis patent atpD 1849 Campylobacter coli This patent atpD 1850Campylobacter fetus subsp. fetus This patent atpD 1851 Campylobacterfetus subsp. venerealis This patent atpD 1852 Campylobacter gracilisThis patent atpD 1853 Campylobacter jejuni subsp. jejuni This patentatpD 1854 Enterococcus cecorum This patent atpD 1855 Enterococcuscolumbae This patent atpD 1856 Enterococcus dispar This patent atpD 1857Enterococcus malodoratus This patent atpD 1858 Enterococcus mundtii Thispatent atpD 1859 Enterococcus raffinosus This patent atpD 1860Globicatella sanguis This patent atpD 1861 Lactococcus garvieae Thispatent atpD 1862 Lactococcus lactis This patent atpD 1863 Listeriaivanovii This patent atpD 1864 Succinivibrio dextrinosolvens This patentatpD 1865 Tetragenococcus halophilus This patent atpD 1866 Campylobacterfetus subsp. fetus This patent recA 1867 Campylobacter fetus subsp.venerealis This patent recA 1868 Campylobacter jejuni subsp. jejuni Thispatent recA 1869 Enterococcus avium This patent recA 1870 Enterococcusfaecium This patent recA 1871 Listeria monocytogenes This patent recA1872 Streptococcus mitis This patent recA 1873 Streptococcus oralis Thispatent recA 1874 Aspergillus fumigatus This patent tuf (M) 1875Aspergillus versicolor This patent tuf (M) 1876 Basidiobolus ranarumThis patent tuf (M) 1877 Campylobacter gracilis This patent tuf 1878Campylobacter jejuni subsp. jejuni This patent tuf 1879 Coccidioidesimmitis This patent tuf (M) 1880 Erwinia amylovora This patent tuf 1881Salmonella choleraesuis subsp. choleraesuis This patent tuf serotypeTyphimurium 1899 Klebsiella pneumoniae Database blaSHV 1900 Klebsiellapneumoniae Database blaSHV 1901 Escherichia coli Database blaSHV 1902Klebsiella pneumoniae Database blaSHV 1903 Klebsiella pneumoniaeDatabase blaSHV 1904 Escherichia coli Database blaSHV 1905 Pseudomonasaeruginosa Database blaSHV 1927 Neisseria meningitidis Database blaTEM1928 Escherichia coli Database blaTEM 1929 Klebsiella oxytoca DatabaseblaTEM 1930 Escherichia coli Database blaTEM 1931 Escherichia coliDatabase blaTEM 1932 Escherichia coli Database blaTEM 1933 Escherichiacoli Database blaTEM 1954 Klebsiella pneumoniae subsp. pneumoniaeDatabase gyrA 1956 Candida inconspicua This patent tuf (M) 1957 Candidautilis This patent tuf (M) 1958 Candida zeylanoides This patent tuf (M)1959 Candida catenulata This patent tuf (M) 1960 Candida krusei Thispatent tuf (M) 1965 Plasmid pGS05 Database sulII 1970 Transposon Tn10Database tetB 1985 Cryptococcus neoformans Database tuf (EF-1) 1986Cryptococcus neoformans Database tuf (EF-1) 1987 Saccharomycescerevisiae Database tuf (EF-1) 1988 Saccharomyces cerevisiae Databasetuf (EF-1) 1989 Eremothecium gossypii Database tuf (EF-1) 1990Eremothecium gossypii Database tuf (EF-1) 1991 Aspergillus oryzaeDatabase tuf (EF-1) 1992 Aureobasidium pullulans Database tuf (EF-1)1993 Histoplasma capsulatum Database tuf (EF-1) 1994 Neurospora crassaDatabase tuf (EF-1) 1995 Podospora anserina Database tuf (EF-1) 1996Podospora curvicolla Database tuf (EF-1) 1997 Sordaria macrosporaDatabase tuf (EF-1) 1998 Trichoderma reesei Database tuf (EF-1) 2004Candida albicans Database tuf (M) 2005 Schizosaccharomyces pombeDatabase tuf (M) 2010 Klebsiella pneumoniae Database blaTEM 2011Klebsiella pneumoniae Database blaTEM 2013 Kluyvera ascorbata Thispatent gyrA 2014 Kluyvera georgiana This patent gyrA 2047 Streptococcuspneumoniae Database pbp1A 2048 Streptococcus pneumoniae Database pbp1A2049 Streptococcus pneumoniae Database pbp1A 2050 Streptococcuspneumoniae Database pbp1A 2051 Streptococcus pneumoniae Database pbp1A2052 Streptococcus pneumoniae Database pbp1A 2053 Streptococcuspneumoniae Database pbp1A 2054 Streptococcus pneumoniae Database gyrA2055 Streptococcus pneumoniae Database parC 2056 Streptococcuspneumoniae This patent pbp1A 2057 Streptococcus pneumoniae This patentpbp1A 2058 Streptococcus pneumoniae This patent pbp1A 2059 Streptococcuspneumoniae This patent pbp1A 2060 Streptococcus pneumoniae This patentpbp1A 2061 Streptococcus pneumoniae This patent pbp1A 2062 Streptococcuspneumoniae This patent pbp1A 2063 Streptococcus pneumoniae This patentpbp1A 2064 Streptococcus pneumoniae This patent pbp1A 2072 Mycobacteriumtuberculosis Database rpoB 2097 Mycoplasma pneumoniae Database tuf 2101Mycobacterium tuberculosis Database inhA 2105 Mycobacterium tuberculosisDatabase embB 2129 Clostridium difficile Database cdtA 2130 Clostridiumdifficile Database cdtB 2137 Pseudomonas putida Genome project tuf 2138Pseudomonas aeruginosa Genome project tuf 2139 Campylobacter jejuniDatabase atpD 2140 Streptococcus pneumoniae Database pbp1a 2144Staphylococcus aureus Database mupA 2147 Escherichia coli Database catI2150 Escherichia coli Database catII 2153 Shigella flexneri DatabasecatIII 2156 Clostridium perfringens Database catP 2159 Staphylococcusaureus Database cat 2162 Staphylococcus aureus Database cat 2165Salmonella typhimurium Database ppflo-like 2183 Alcaligenes faecalissubsp. faecalis This patent tuf 2184 Campylobacter coli This patent fusA2185 Succinivibrio dextrinosolvens This patent tuf 2186 Tetragenococcushalophilus This patent tuf 2187 Campylobacter jejuni subsp. jejuni Thispatent fusA 2188 Campylobacter jejuni subsp. jejuni This patent fusA2189 Leishmania guyanensis This patent atpD 2190 Trypanosoma bruceibrucei This patent atpD 2191 Aspergillus nidulans This patent atpD 2192Leishmania panamensis This patent atpD 2193 Aspergillus nidulans Thispatent tuf (M) 2194 Aureobasidium pullulans This patent tuf (M) 2195Emmonsia parva This patent tuf (M) 2196 Exserohilum rostratum Thispatent tuf (M) 2197 Fusarium moniliforme This patent tuf (M) 2198Fusarium solani This patent tuf (M) 2199 Histoplasma capsulatum Thispatent tuf (M) 2200 Kocuria kristinae This patent tuf 2201 Vibriomimicus This patent tuf 2202 Citrobacter freundii This patent recA 2203Clostridium botulinum This patent recA 2204 Francisella tularensis Thispatent recA 2205 Peptostreptococcus anaerobius This patent recA 2206Peptostreptococcus asaccharolyticus This patent recA 2207 Providenciastuartii This patent recA 2208 Salmonella choleraesuis subsp.choleraesuis This patent recA serotype Paratyphi A 2209 Salmonellacholeraesuis subsp. choleraesuis This patent recA serotype Typhimurium2210 Staphylococcus saprophyticus This patent recA 2211 Yersiniapseudotuberculosis This patent recA 2212 Zoogloea ramigera This patentrecA 2214 Abiotrophia adiacens This patent fusA 2215 Acinetobacterbaumannii This patent fusA 2216 Actinomyces meyeri This patent fusA 2217Clostridium difficile This patent fusA 2218 Corynebacterium diphtheriaeThis patent fusA 2219 Enterobacter cloacae This patent fusA 2220Klebsiella pneumoniae subsp. pneumoniae This patent fusA 2221 Listeriamonocytogenes This patent fusA 2222 Mycobacterium avium This patent fusA2223 Mycobacterium gordonae This patent fusA 2224 Mycobacterium kansasiiThis patent fusA 2225 Mycobacterium terrae This patent fusA 2226Neisseria polysaccharea This patent fusA 2227 Staphylococcus epidermidisThis patent fusA 2228 Staphylococcus haemolyticus This patent fusA 2229Succinivibrio dextrinosolvens This patent fusA 2230 Tetragenococcushalophilus This patent fusA 2231 Veillonella parvula This patent fusA2232 Yersinia pseudotuberculosis This patent fusA 2233 Zoogloea ramigeraThis patent fusA 2234 Aeromonas hydrophila This patent fusA 2235Abiotrophia adiacens This patent fusA-tuf spacer 2236 Acinetobacterbaumannii This patent fusA-tuf spacer 2237 Actinomyces meyeri Thispatent fusA-tuf spacer 2238 Clostridium difficile This patent fusA-tufspacer 2239 Corynebacterium diphtheriae This patent fusA-tuf spacer 2240Enterobacter cloacae This patent fusA-tuf spacer 2241 Klebsiellapneumoniae subsp. pneumoniae This patent fusA-tuf spacer 2242 Listeriamonocytogenes This patent fusA-tuf spacer 2243 Mycobacterium avium Thispatent fusA-tuf spacer 2244 Mycobacterium gordonae This patent fusA-tufspacer 2245 Mycobacterium kansasii This patent fusA-tuf spacer 2246Mycobacterium terrae This patent fusA-tuf spacer 2247 Neisseriapolysaccharea This patent fusA-tuf spacer 2248 Staphylococcusepidermidis This patent fusA-tuf spacer 2249 Staphylococcus haemolyticusThis patent fusA-tuf spacer 2255 Abiotrophia adiacens This patent tuf2256 Acinetobacter baumannii This patent tuf 2257 Actinomyces meyeriThis patent tuf 2258 Clostridium difficile This patent tuf 2259Corynebacterium diphtheriae This patent tuf 2260 Enterobacter cloacaeThis patent tuf 2261 Klebsiella pneumoniae subsp. pneumoniae This patenttuf 2262 Listeria monocytogenes This patent tuf 2263 Mycobacterium aviumThis patent tuf 2264 Mycobacterium gordonae This patent tuf 2265Mycobacterium kansasii This patent tuf 2266 Mycobacterium terrae Thispatent tuf 2267 Neisseria polysaccharea This patent tuf 2268Staphylococcus epidermidis This patent tuf 2269 Staphylococcushaemolyticus This patent tuf 2270 Aeromonas hydrophila This patent tuf2271 Bilophila wadsworthia This patent tuf 2272 Brevundimonas diminutaThis patent tuf 2273 Streptococcus mitis This patent pbp1a 2274Streptococcus mitis This patent pbp1a 2275 Streptococcus mitis Thispatent pbp1a 2276 Streptococcus oralis This patent pbp1a 2277Escherichia coli This patent gyrA 2278 Escherichia coli This patent gyrA2279 Escherichia coli This patent gyrA 2280 Escherichia coli This patentgyrA 2288 Enterococcus faecium Database ddl 2293 Enterococcus faeciumDatabase vanA 2296 Enterococcus faecalis Database vanB *tuf indicatestuf sequences, tuf (C) indicates tuf sequences divergent from main(usually A and B) copies of the elongation factor-Tu, tuf (EF-1)indicates tuf sequences of the eukaryotic type (elongation factor 1α),tuf (M) indicates tuf sequences from organellar (mostly mitochondrial)origin. fusA indicates fusA sequences; fusA-tuf spacer indicates theintergenic region between fusA and tuf. atpD indicates atpD sequences ofthe F-type, atpD (V) indicates atpD sequences of the V-type. recAindicates recA sequences, recA(Rad51) indicates rad51 sequences orhomologs and recA(Dmc1) indicates dmc1 sequences or homologs.

TABLE 8 Bacterial species used to test the specificity of theStreptococcus agalactiae-specific amplification primers derived from tufsequences. Strain Reference number Streptococcus acidominimus ATCC 51726Streptococcus agalactiae ATCC 12403 Streptococcus agalactiae ATCC 12973Streptococcus agalactiae ATCC 13813 Streptococcus agalactiae ATCC 27591Streptococcus agalactiae CDCs 1073 Streptococcus anginosus ATCC 27335Streptococcus anginosus ATCC 33397 Streptococcus bovis ATCC 33317Streptococcus anginosus ATCC 27823 Streptococcus cricetus ATCC 19642Streptococcus cristatus ATCC 51100 Streptococcus downei ATCC 33748Streptococcus dysgalactiae ATCC 43078 Streptococcus equi subsp. equiATCC 9528 Streptococcus ferus ATCC 33477 Streptococcus gordonii ATCC10558 Streptococcus macacae ATCC 35911 Streptococcus mitis ATCC 49456Streptococcus mutans ATCC 25175 Streptococcus oralis ATCC 35037Streptococcus parasanguinis ATCC 15912 Streptococcus parauberis DSM 6631Streptococcus pneumoniae ATCC 27336 Streptococcus pyogenes ATCC 19615Streptococcus ratti ATCC 19645 Streptococcus salivarius ATCC 7073Streptococcus sanguinis ATCC 10556 Streptococcus sobrinus ATCC 27352Streptococcus suis ATCC 43765 Streptococcus uberis ATCC 19436Streptococcus vestubularis ATCC 49124 Bacteroides caccae ATCC 43185Bacteroides vulgatus ATCC 8482 Bacteroides fragilis ATCC 25285 Candidaalbicans ATCC 11006 Clostridium innoculum ATCC 14501 Clostridium ramosumATCC 25582 Lactobacillus casei subsp. casei ATCC 393 Clostridiumsepticum ATCC 12464 Corynebacterium cervicis NCTC 10604 Corynebacteriumgenitalium ATCC 33031 Corynebacterium urealyticum ATCC 43042Enterococcus faecalis ATCC 29212 Enterococcus faecium ATCC 19434Eubacterium lentum ATCC 43055 Eubacterium nodutum ATCC 33099 Gardnerellavaginalis ATCC 14018 Lactobacillus acidophilus ATCC 4356 Lactobacilluscrispatus ATCC 33820 Lactobacillus gasseri ATCC 33323 Lactobacillusjohnsonii ATCC 33200 Lactococcus lactis subsp. lactis ATCC 19435Lactococcus lactis subsp. lactis ATCC 11454 Listeria innocua ATCC 33090Micrococcus luteus ATCC 9341 Escherichia coli ATCC 25922 Micrococcuslylae ATCC 27566 Porphyromonas asaccharolytica ATCC 25260 Prevotellacorporis ATCC 33547 Prevotella melanogenica ATCC 25845 Staphylococcusaureus ATCC 13301 Staphylococcus epidermidis ATCC 14990 Staphylococcussaprophyticus ATCC 15305

TABLE 9 Bacterial species used to test the specificity of theStreptococcus agalactiae-specific amplification primers derived fromatpD sequences. Strain Reference number Streptococcus acidominimus ATCC51726 Streptococcus agalactiae ATCC 12400 Streptococcus agalactiae ATCC12403 Streptococcus agalactiae ATCC 12973 Streptococcus agalactiae ATCC13813 Streptococcus agalactiae ATCC 27591 Streptococcus agalactiaeCDCs-1073 Streptococcus anginosus ATCC 27335 Streptococcus anginosusATCC 27823 Streptococcus bovis ATCC 33317 Streptococcus cricetus ATCC19642 Streptococcus cristatus ATCC 51100 Streptococcus downei ATCC 33748Streptococcus dysgalactiae ATCC 43078 Streptococcus equi subsp. equiATCC 9528 Streptococcus ferus ATCC 33477 Streptococcus gordonii ATCC10558 Streptococcus macacae ATCC 35911 Streptococcus mitis ATCC 49456Streptococcus mutans ATCC 25175 Streptococcus oralis ATCC 35037Streptococcus parasanguinis ATCC 15912 Streptococcus parauberis DSM 6631Streptococcus pneumoniae ATCC 27336 Streptococcus pyogenes ATCC 19615Streptococcus ratti ATCC 19645 Streptococcus salivarius ATCC 7073Streptococcus sanguinis ATCC 10556 Streptococcus sobrinus ATCC 27352Streptococcus suis ATCC 43765 Streptococcus uberis ATCC 19436Streptococcus vestibularis ATCC 49124

TABLE 10 Bacterial species used to test the specificity of theEnterococcus-specific amplification primers derived from tuf sequences.Strain Reference number Gram-positive species (n = 74) Abiotrophiaadiacens ATCC 49176 Abiotrophia defectiva ATCC 49175 Bacillus cereusATCC 14579 Bacillus subtilis ATCC 27370 Bifidobacterium adolescentisATCC 27534 Bifidobacterium breve ATCC 15700 Bifidobacterium dentium ATCC27534 Bifidobacterium longum ATCC 15707 Clostridium perfringens ATCC3124 Clostridium septicum ATCC 12464 Corynebacterium aquaticus ATCC14665 Corynebacterium pseudodiphtheriticum ATCC 10700 Enterococcus aviumATCC 14025 Enterococcus casseliflavus ATCC 25788 Enterococcus cecorumATCC 43199 Enterococcus columbae ATCC 51263 Enterococcus dispar ATCC51266 Enterococcus durans ATCC 19432 Enterococcus faecalis ATCC 29212Enterococcus faecium ATCC 19434 Enterococcus flavescens ATCC 49996Enterococcus gallinarum ATCC 49573 Enterococcus hirae ATCC 8044Enterococcus malodoratus ATCC 43197 Enterococcus mundtii ATCC 43186Enterococcus pseudoavium ATCC 49372 Enterococcus raffinosus ATCC 49427Enterococcus saccharolyticus ATCC 43076 Enterococcus solitarius ATCC49428 Enterococcus sulfureus ATCC 49903 Eubacterium lentum ATCC 49903Gemella haemolysans ATCC 10379 Gemella morbillorum ATCC 27842Lactobacillus acidophilus ATCC 4356 Leuconostoc mesenteroides ATCC 19225Listeria grayi ATCC 19120 Listeria grayi ATCC 19123 Listeria innocuaATCC 33090 Listeria ivanovii ATCC 19119 Listeria monocytogenes ATCC15313 Listeria seeligeri ATCC 35967 Micrococcus luteus ATCC 9341Pediococcus acidilacti ATCC 33314 Pediococcus pentosaceus ATCC 33316Peptococcus niger ATCC 27731 Peptostreptococcus anaerobius ATCC 27337Peptostreptococcus indolicus ATCC 29247 Peptostreptococcus micros ATCC33270 Propionibacterium acnes ATCC 6919 Staphylococcus aureus ATCC 43300Staphylococcus capitis ATCC 27840 Staphylococcus epidermidis ATCC 14990Staphylococcus haemolyticus ATCC 29970 Staphylococcus hominis ATCC 27844Staphylococcus lugdunensis ATCC 43809 Staphylococcus saprophyticus ATCC15305 Staphylococcus simulans ATCC 27848 Staphylococcus warneri ATCC27836 Streptococcus agalactiae ATCC 13813 Streptococcus anginosus ATCC33397 Streptococcus bovis ATCC 33317 Streptococcus constellatus ATCC27823 Streptococcus cristatus ATCC 51100 Streptococcus intermedius ATCC27335 Streptococcus mitis ATCC 49456 Streptococcus mitis ATCC 3639Streptococcus mutans ATCC 27175 Streptococcus parasanguinis ATCC 15912Streptococcus pneumoniae ATCC 27736 Streptococcus pneumoniae ATCC 6303Streptococcus pyogenes ATCC 19615 Streptococcus salivarius ATCC 7073Streptococcus sanguinis ATCC 10556 Streptococcus suis ATCC 43765Gram-negative species (n = 39) Acidominococcus fermentans ATCC 2508Acinetobacter baumannii ATCC 19606 Alcaligenes faecalis ATCC 8750Anaerobiospirillum ATCC 29305 succiniproducens Anaerorhabdus furcosusATCC 25662 Bacteroides distasonis ATCC 8503 Bacteroides thetaiotaomicronATCC 29741 Bacteroides vulgatus ATCC 8482 Bordetella pertussis LSPQ 3702Bulkholderia cepacia LSPQ 2217 Butyvibrio fibrinosolvens ATCC 19171Cardiobacterium hominis ATCC 15826 Citrobacter freundii ATCC 8090Desulfovibrio vulgaris ATCC 29579 Edwardsiellae tarda ATCC 15947Enterobacter cloacae ATCC 13047 Escherichia coli ATCC 25922Fusobacterium russii ATCC 25533 Haemophilus influenzae ATCC 9007 Hafniaalvei ATCC 13337 Klebsiella oxytoca ATCC 13182 Meganomonas hypermegasATCC 25560 Mitsukoella multiacidus ATCC 27723 Moraxella catarrhalis ATCC43628 Morganella morganii ATCC 25830 Neisseria meningitidis ATCC 13077Pasteurella aerogenes ATCC 27883 Proteus vulgaris ATCC 13315 Providenciaalcalifaciens ATCC 9886 Providencia rettgeri ATCC 9250 Pseudomonasaeruginosa ATCC 27853 Salmonella typhimurium ATCC 14028 Serratiamarcescens ATCC 13880 Shigella flexneri ATCC 12022 Shigella sonnei ATCC29930 Succinivibrio dextrinosolvens ATCC 19716 Tissierella praeacutaATCC 25539 Veillonella parvuala ATCC 10790 Yersinia enterocolitica ATCC9610

TABLE 11 Microbial species for which tuf and/or atpD and/or recAsequences are available in public databases. Species Strain Accessionnumber Coding gene* tuf sequences Bacteria Actinobacillusactinomycetemcomitans HK1651 Genome project² tuf Actinobacillusactinomycetemcomitans HK1651 Genome project² tuf (EF-G) Agrobacteriumtumefaciens X99673 tuf Agrobacterium tumefaciens X99673 tuf (EF-G)Agrobacterium tumefaciens X99674 tuf Anacystis nidulans PCC 6301 X17442tuf Aquifex aeolicus VF5 AE000669 tuf Aquifex aeolicus VF5 AE000669 tuf(EF-G) Aquifex pyrophilus Genome project² tuf (EF-G) Aquifex pyrophilusY15787 tuf Bacillus anthracis Ames Genome project² tuf Bacillusanthracis Ames Genome project² tuf (EF-G) Bacillus halodurans C-125AB017508 tuf Bacillus halodurans C-125 AB017508 tuf (EF-G) Bacillusstearothermophilus CCM 2184 AJ000260 tuf Bacillus subtilis 168 D64127tuf Bacillus subtilis 168 D64127 tuf (EF-G) Bacillus subtilis DSM 10Z99104 tuf Bacillus subtilis DSM 10 Z99104 tuf (EF-G) Bacteroidesforsythus ATCC 43037 AB035466 tuf Bacteroides fragilis DSM 1151 —¹ tufBordetella bronchiseptica RB50 Genome project² tuf Bordetella pertussisTohama 1 Genome project² tuf Bordetella pertussis Tohama 1 Genomeproject² tuf (EF-G) Borrelia burdorgferi B31 U78193 tuf Borreliaburgdorferi AE001155 tuf (EF-G) Brevibacterium linens DSM 20425 X76863tuf Buchnera aphidicola Ap Y12307 tuf Burkholderia pseudomallei K96243Genome project² tuf (EF-G) Campylobacter jejuni NCTC 11168 Y17167 tufCampylobacter jejuni NCTC 11168 CJ11168X2 tuf (EF-G) Chlamydiapneumoniae CWL029 AE001592 tuf Chlamydia pneumoniae CWL029 AE001639 tuf(EF-G) Chlamydia trachomatis M74221 tuf Chlamydia trachomatis D/UW-3/CXAE001317 tuf (EF-G) Chlamydia trachomatis D/UW-3/CX AE001305 tufChlamydia trachomatis F/IC-Cal-13 L22216 tuf Chlorobium vibrioforme DSM263 X77033 tuf Chloroflexus aurantiacus DSM 636 X76865 tuf Clostridiumacetobutylicum ATCC 824 Genome project² tuf Clostridium difficile 630Genome project² tuf Clostridium difficile 630 Genome project² tuf (EF-G)Corynebacterium diphtheriae NCTC 13129 Genome project² tufCorynebacterium diphtheriae NCTC 13129 Genome project² tuf (EF-G)Corynebacterium glutamicum ASO 19 X77034 tuf Corynebacterium glutamicumMJ-233 E09634 tuf Coxiella burnetii Nine Mile phase I AF136604 tufCytophaga lytica DSM 2039 X77035 tuf Deinococcus radiodurans R1 AE001891tuf (EF-G) Deinococcus radiodurans R1 AE180092 tuf Deinococcusradiodurans R1 AE002041 tuf Deinonema sp. —¹ tuf Eikenella corrodensATCC 23834 Z12610 tuf Eikenella corrodens ATCC 23834 Z12610 tuf (EF-G)Enterococcus faecalis Genome project² tuf (EF-G) Escherichia coli J01690tuf Escherichia coli J01717 tuf Escherichia coli X00415 tuf (EF-G)Escherichia coli X57091 tuf Escherichia coli K-12 MG1655 U00006 tufEscherichia coli K-12 MG1655 U00096 tuf Escherichia coli K-12 MG1655AE000410 tuf (EF-G) Fervidobacterium islandicum DSM 5733 Y15788 tufFibrobacter succinogenes S85 X76866 tuf Flavobacterium ferrigeneum DSM13524 X76867 tuf Flexistipes sinusarabici X59461 tuf Gloeobacterviolaceus PCC 7421 U09433 tuf Gloeothece sp. PCC 6501 U09434 tufHaemophilus actinomycetemcomitans HK1651 Genome project² tuf Haemophilusducreyi 35000 AF087414 tuf (EF-G) Haemophilus influenzae Rd U32739 tufHaemophilus influenzae Rd U32746 tuf Haemophilus influenzae Rd U32739tuf (EF-G) Helicobacter pylori 26695 AE000511 tuf Helicobacter pyloriJ99 AE001539 tuf (EF-G) Helicobacter pylori J99 AE001541 tufHerpetosiphon aurantiacus Hpga1 X76868 tuf Klebsiella pneumoniae M6H78578 Genome project² tuf Klebsiella pneumoniae M6H 78578 Genomeproject² tuf (EF-G) Lactobacillus paracasei E13922 tuf Legionellapneumophila Philadelphia-1 Genome project² tuf Leptospira interrogansAF115283 tuf Leptospira interrogans AF115283 tuf (EF-G) Micrococcusluteus IFO 3333 M17788 tuf (EF-G) Micrococcus luteus IFO 3333 M17788 tufMoraxella sp. TAC II 25 AJ249258 tuf Mycobacterium avium 104 Genomeproject² tuf Mycobacterium avium 104 Genome project² tuf (EF-G)Mycobacterium bovis AF2122/97 Genome project² tuf Mycobacterium bovisAF2122/97 Genome project² tuf (EF-G) Mycobacterium leprae L13276 tufMycobacterium leprae Z14314 tuf Mycobacterium leprae Z14314 tuf (EF-G)Mycobacterium leprae Thai 53 D13869 tuf Mycobacterium tuberculosisErdmann S40925 tuf Mycobacterium tuberculosis H37Rv AL021943 tuf (EF-G)Mycobacterium tuberculosis H37Rv Z84395 tuf Mycobacterium tuberculosisy42 AD000005 tuf Mycobacterium tuberculosis CSU#93 Genome project² tufMycobacterium tuberculosis CSU#93 Genome project² tuf (EF-G) Mycoplasmacapricolum PG-31 X16462 tuf Mycoplasma genitalium G37 U39732 tufMycoplasma genitalium G37 U39689 tuf (EF-G) Mycoplasma hominis X57136tuf Mycoplasma hominis PG21 M57675 tuf Mycoplasma pneumoniae M129AE000019 tuf Mycoplasma pneumoniae M129 AE000058 tuf (EF-G) Neisseriagonorrhoeae MS11 L36380 tuf Neisseria gonorrhoeae MS11 L36380 tuf (EF-G)Neisseria meningitidis Z2491 Genome project² tuf (EF-G) Neisseriameningitidis Z2491 Genome project² tuf Pasteurella multocida Pm70 Genomeproject² tuf Peptococcus niger DSM 20745 X76869 tuf Phormidium ectocarpiPCC 7375 U09443 tuf Planobispora rosea ATCC 53773 U67308 tufPlanobispora rosea ATCC 53733 X98830 tuf Planobispora rosea ATCC 53733X98830 tuf (EF-G) Plectonema boryanum PCC 73110 U09444 tuf Porphyromonasgingivalis W83 Genome project² tuf Porphyromonas gingivalis W83 Genomeproject² tuf (EF-G) Porphyromonas gingivalis FDC 381 AB035461 tufPorphyromonas gingivalis W83 AB035462 tuf Porphyromonas gingivalis SUNY1021 AB035463 tuf Porphyromonas gingivalis A7A1-28 AB035464 tufPorphyromonas gingivalis ATCC 33277 AB035465 tuf Porphyromonasgingivalis ATCC 33277 AB035471 tuf (EF-G) Prochlorothrix hollandicaU09445 tuf Pseudomonas aeruginosa PAO-1 Genome project² tuf Pseudomonasputida Genome project² tuf Rickettsia prowazekii Madrid E AJ235272 tufRickettsia prowazekii Madrid E AJ235270 tuf (EF-G) Rickettsia prowazekiiMadrid E Z54171 tuf (EF-G) Salmonella choleraesuis subsp. X64591 tuf(EF-G) choleraesuis serotype Typhimurium Salmonella choleraesuis subsp.LT2 trpE91 X55116 tuf choleraesuis serotype Typhimurium Salmonellacholeraesuis subsp. LT2 trpE91 X55117 tuf choleraesuis serotypeTyphimurium Serpulina hyodysenteriae B204 U51635 tuf Serratia marcescensAF058451 tuf Shewanella putrefaciens DSM 50426 —¹ tuf Shewanellaputrefaciens MR-1 Genome project² tuf Spirochaeta aurantia DSM 1902X76874 tuf Staphylococcus aureus AJ237696 tuf (EF-G) Staphylococcusaureus EMRSA-16 Genome project² tuf Staphylococcus aureus NCTC 8325Genome project² tuf Staphylococcus aureus COL Genome project² tufStaphylococcus aureus EMRSA-16 Genome project² tuf (EF-G) Stigmatellaaurantiaca DW4 X82820 tuf Stigmatella aurantiaca Sg a1 X76870 tufStreptococcus mutans GS-5 Kuramitsu U75481 tuf Streptococcus mutansUAB159 Genome project² tuf Streptococcus oralis NTCC 11427 P331701 tufStreptococcus pyogenes Genome project² tuf (EF-G) Streptococcus pyogenesM1-GAS Genome project² tuf Streptomyces aureofaciens ATCC 10762 AF007125tuf Streptomyces cinnamoneus Tue89 X98831 tuf Streptomyces coelicolorA3(2) AL031013 tuf (EF-G) Streptomyces coelicolor A3(2) X77039 tuf(EF-G) Streptomyces coelicolor M145 X77039 tuf Streptomyces collinus BSM40733 S79408 tuf Streptomyces netropsis Tu1063 AF153618 tuf Streptomycesramocissimus X67057 tuf Streptomyces ramocissimus X67058 tufStreptomyces ramocissimus X67057 tuf (EF-G) Synechococcus sp. PCC 6301X17442 tuf (EF-G) Synechococcus sp. PCC 6301 X17442 tuf Synechocystissp. PCC 6803 D90913 tuf (EF-G) Synechocystis sp. PCC 6803 D90913 tufSynechocystis sp. PCC 6803 X65159 tuf (EF-G) Taxeobacter occealus Myx2105 X77036 tuf Thermotoga maritima Genome project² tuf (EF-G)Thermotoga maritima M27479 tuf Thermus aquaticus EP 00276 X66322 tufThermus thermophilus HB8 X16278 tuf (EF-G) Thermus thermophilus HB8X05977 tuf Thermus thermophilus HB8 X06657 tuf Thiomonas cuprina DSM5495 U78300 tuf Thiomonas cuprina DSM 5495 U78300 tuf (EF-G) Thiomonascuprina Hoe5 X76871 tuf Treponema denticola Genome project² tufTreponema denticola Genome project² tuf (EF-G) Treponema pallidumAE001202 tuf Treponema pallidum AE001222 tuf (EF-G) Treponema pallidumAE001248 tuf (EF-G) Ureaplasma urealyticum ATCC 33697 Z34275 tufUreaplasma urealyticum serovar 3 biovar 1 AE002151 tuf Ureaplasmaurealyticum serovar 3 biovar 1 AE002151 tuf (EF-G) Vibrio choleraeN16961 Genome project² tuf Wolinella succinogenes DSM 1740 X76872 tufYersinia pestis CO-92 Genome project² tuf Yersinia pestis CO-92 Genomeproject² tuf (EF-G) Archaebacteria Archaeoglobus fulgidus Genomeproject² tuf (EF-G) Halobacterium marismortui X16677 tufMethanobacterium thermoautrophicum delta H AE000877 tuf Methanococcusjannaschii ATCC 43067 U67486 tuf Methanococcus vannielii X05698 tufPyrococcus abyssi Orsay AJ248285 tuf Thermoplasma acidophilum DSM 1728X53866 tuf Fungi Absidia glauca CBS 101.48 X54730 tuf (EF-1) Arxulaadeninivorans Ls3 Z47379 tuf (EF-1) Aspergillus oryzae KBN616 AB007770tuf (EF-1) Aureobasidium pullulans R106 U19723 tuf (EF-1) Candidaalbicans SC5314 Genome project² tuf (M) Candida albicans SC5314 M29934tuf (EF-1) Candida albicans SC5314 M29935 tuf (EF-1) Cryptococcusneoformans B3501 U81803 tuf (EF-1) Cryptococcus neoformans M1-106 U81804tuf (EF-1) Eremothecium gossypii ATCC 10895 X73978 tuf (EF-1)Eremothecium gossypii A29820 tuf (EF-1) Fusarium oxysporum NRRL 26037AF008498 tuf (EF-1) Histoplasma capsulatum 186AS U14100 tuf (EF-1)Podospora anserina X74799 tuf (EF-1) Podospora curvicolla VLV X96614 tuf(EF-1) Prototheca wickerhamii 263-11 AJ245645 tuf (EF-1) Pucciniagraminis race 32 X73529 tuf (EF-1) Reclinomonas americana ATCC 50394AF007261 tuf (M) Rhizomucor racemosus ATCC 1216B X17475 tuf (EF-1)Rhizomucor racemosus ATCC 1216B J02605 tuf (EF-1) Rhizomucor racemosusATCC 1216B X17476 tuf (EF-1) Rhodotorula mucilaginosa AF016239 tuf(EF-1) Saccharomyces cerevisiae K00428 tuf (M) Saccharomyces cerevisiaeM59369 tuf (EF-G) Saccharomyces cerevisiae X00779 tuf (EF-1)Saccharomyces cerevisiae X01638 tuf (EF-1) Saccharomyces cerevisiaeM10992 tuf (EF-1) Saccharomyces cerevisiae Alpha S288 X78993 tuf (EF-1)Saccharomyces cerevisiae M15666 tuf (EF-1) Saccharomyces cerevisiaeZ35987 tuf (EF-1) Saccharomyces cerevisiae S288C (AB972) U51033 tuf(EF-1) Schizophyllum commune 1-40 X94913 tuf (EF-1) Schizosaccharomycespombe 972h- AL021816 tuf (EF-1) Schizosaccharomyces pombe 972h- AL021813tuf (EF-1) Schizosaccharomyces pombe 972h- D82571 tuf (EF-1)Schizosaccharomyces pombe U42189 tuf (EF-1) Schizosaccharomyces pombePR745 D89112 tuf (EF-1) Sordaria macrospora OOO X96615 tuf (EF-1)Trichoderma reesei QM9414 Z23012 tuf (EF-1) Yarrowia lipolytica AF054510tuf (EF-1) Parasites Blastocystis hominis HE87-1 D64080 tuf (EF-1)Cryptosporidium parvum U69697 tuf (EF-1) Eimeria tenella LS18 AI755521tuf (EF-1) Entamoeba histolytica HM1:IMSS X83565 tuf (EF-1) Entamoebahistolytica NIH 200 M92073 tuf (EF-1) Giardia lamblia D14342 tuf (EF-1)Kentrophoros sp. AF056101 tuf (EF-1) Leishmania amazonensisIFLA/BR/67/PH8 M92653 tuf (EF-1) Leishmania braziliensis U72244 tuf(EF-1) Onchocerca volvulus M64333 tuf (EF-1) Porphyra purpurea AvonportU08844 tuf (EF-1) Plasmodium berghei ANKA AJ224150 tuf (EF-1) Plasmodiumfalciparum K1 X60488 tuf (EF-1) Plasmodium knowlesi line H AJ224153 tuf(EF-1) Toxoplasma gondii RH Y11431 tuf (EF-1) Trichomonas tenax ATCC30207 D78479 tuf (EF-1) Trypanosoma brucei LVH/75/ U10562 tuf (EF-1)USAMRU-K/18 Trypanosoma cruzi Y L76077 tuf (EF-1) Human and plantsArabidopsis thaliana Columbia X89227 tuf (EF-1) Glycine max CeresiaX89058 tuf (EF-1) Glycine max Ceresia Y15107 tuf (EF-1) Glycine maxCeresia Y15108 tuf (EF-1) Glycine max Maple Arrow X66062 tuf (EF-1) Homosapiens X03558 tuf (EF-1) Pyramimonas disomata AB008010 tuf atpDsequences Bacteria Acetobacterium woodi DSM 1030 U10505 atpDActinobacillus actinomycetemcomitans HK1651 Genome project² atpDBacillus anthracis Ames Genome project² atpD Bacillus firmus OF4 M60117atpD Bacillus megaterium QM B1551 M20255 atpD Bacillusstearothermophilus D38058 atpD Bacillus stearothermophilus IFO1035D38060 atpD Bacillus subtilis 168 Z28592 atpD Bacteroides fragilis DSM2151 M22247 atpD Bordetella bronchiseptica RB50 Genome project² atpDBordetella pertussis Tohama 1 Genome project² atpD Borrelia burgdorferiB31 AE001122 atpD (V) Burkholderia cepacia DSM50181 X76877 atpDBurkholderia pseudomallei K96243 Genome project² atpD Campylobacterjejuni NCTC 11168 CJ11168X1 atpD Chlamydia pneumoniae Genome project²atpD (V) Chlamydia trachomatis MoPn Genome project² atpD (V) Chlorobiumvibrioforme DSM 263 X76873 atpD Citrobacter freundii JEO503 AF037156atpD Clostridium acetobutylicum ATCC 824 Genome project² atpDClostridium acetobutylicum DSM 792 AF101055 atpD Clostridium difficile630 Genome project² atpD Corynebacterium diphtheriae NCTC13129 Genomeproject² atpD Corynebacterium glutamicum ASO 19 X76875 atpDCorynebacterium glutamicum MJ-233 E09634 atpD Cytophaga lytica DSM 2039M22535 atpD Enterobacter aerogenes DSM 30053 —³ atpD Enterococcusfaecalis V583 Genome project² atpD (V) Enterococcus hirae M90060 atpDEnterococcus hirae ATCC 9790 D17462 atpD (V) Escherichia coli J01594atpD Escherichia coli M25464 atpD Escherichia coli V00267 atpDEscherichia coli V00311 atpD Escherichia coli K12 MG1655 L10328 atpDFlavobacterium ferrugineum DSM 13524 —³ atpD Haemophilusactinomycetemcomitans Genome project² atpD Haemophilus influenzae RdU32730 atpD Helicobacter pylori NCTC 11638 AF004014 atpD Helicobacterpylori 26695 Genome project² atpD Helicobacter pylori J99 Genomeproject² atpD Klebsiella pneumoniae M6H 78578 Genome project² atpDLactobacillus casei DSM 20021 X64542 atpD Legionella pneumophilaPhiladelphia-1 Genome project² atpD Moorella thermoacetica ATCC 39073U64318 atpD Mycobacterium avium 104 Genome project² atpD Mycobacteriumbovis AF2122/97 Genome project² atpD Mycobacterium leprae U15186 atpDMycobacterium leprae Genome project² atpD Mycobacterium tuberculosisH37Rv Z73419 atpD Mycobacterium tuberculosis CSU#93 Genome project² atpDMycoplasma gallisepticum X64256 atpD Mycoplasma genitalium G37 U39725atpD Mycoplasma pneumoniae M129 U43738 atpD Neisseria gonorrhoeae FA1090 Genome project² atpD Neisseria meningitidis Z2491 Genome project²atpD Pasteurella multocida Pm70 Genome project² atpD Pectinatusfrisingensis DSM 20465 X64543 atpD Peptococcus niger DSM 20475 X76878atpD Pirellula marina IFAM 1313 X57204 atpD Porphyromonas gingivalis W83Genome project² atpD (V) Propionigenium modestum DSM 2376 X58461 atpDPseudomonas aeruginosa PAO1 Genome project² atpD Pseudomonas putidaGenome project² atpD Rhodobacter capsulatus B100 X99599 atpDRhodospirillum rubrum X02499 atpD Rickettsia prowazekii F-12 AF036246atpD Rickettsia prowazekii Madrid Genome project² atpD Ruminococcusalbus 7ATCC AB006151 atpD Salmonella bongori JEO4162 AF037155 atpDSalmonella bongori BR1859 AF037154 atpD Salmonella choleraesuis S83769AF037146 atpD subsp. arizonae Salmonella choleraesuis u24 AF037147 atpDsubsp. arizonae Salmonella choleraesuis subsp. K228 AF037140 atpDcholeraesuis serotype Dublin Salmonella choleraesuis subsp. K771AF037139 atpD choleraesuis serotype Dublin Salmonella choleraesuissubsp. Div36-86 AF037142 atpD choleraesuis serotype Infantis Salmonellacholeraesuis subsp. Div95-86 AF037143 atpD choleraesuis serotypeTennessee Salmonella choleraesuis subsp. LT2 AF037141 atpD choleraesuisserotype Typhimurium Salmonella choleraesuis DS210/89 AF037149 atpDsubsp. diarizonae Salmonella choleraesuis JEO307 AF037148 atpD subsp.diarizonae Salmonella choleraesuis S109671 AF037150 atpD subsp.diarizonae Salmonella choleraesuis S84366 AF037151 atpD subsp. houtenaeSalmonella choleraesuis S84098 AF037152 atpD subsp. houtenae Salmonellacholeraesuis BR2047 AF037153 atpD subsp. indica Salmonella choleraesuisNSC72 AF037144 atpD subsp. salamae Salmonella choleraesuis S114655AF037145 atpD subsp. salamae Shewanella putrefaciens MR-1 Genomeproject² atpD Staphylococcus aureus COL Genome project² atpD Stigmatellaaurantiaca Sga1 X76879 atpD Streptococcus bovis JB-1 AB009314 atpDStreptococcus mutans GS-5 U31170 atpD Streptococcus mutans UAB159 Genomeproject² atpD Streptococcus pneumoniae Type 4 Genome project² atpD (V)Streptococcus pneumoniae Type 4 Genome project² atpD Streptococcuspyogenes M1-GAS Genome project² atpD (V) Streptococcus pyogenes M1-GASGenome project² atpD Streptococcus sanguinis 10904 AF001955 atpDStreptomyces lividans 1326 Z22606 atpD Thermus thermophilus HB8 D63799atpD (V) Thiobacillus ferrooxidans ATCC 33020 M81087 atpD Treponemapallidum Nichols AE001228 atpD (V) Vibrio alginolyticus X16050 atpDVibrio cholerae N16961 Genome project² atpD Wolinella succinogenes DSM1470 X76880 atpD Yersinia enterocolitica NCTC 10460 AF037157 atpDYersinia pestis CO-92 Genome project² atpD Archaebacteria Archaeoglobusfulgidus DSM 4304 AE001023 atpD (V) Halobacterium salinarum S56356 atpD(V) Haloferax volcanii WR 340 X79516 atpD Methanococcus jannaschii DSM2661 U67477 atpD (V) Methanosarcina barkeri DSM 800 J04836 atpD (V)Fungi Candida albicans SC5314 Genome project² atpD Candida tropicalisM64984 atpD (V) Kluyveromyces lactis 2359/152 U37764 atpD Neurosporacrassa X53720 atpD Saccharomyces cerevisiae M12082 atpD Saccharomycescerevisiae X2180-1A J05409 atpD (V) Schizosaccharomyces pombe 972 h-S47814 atpD (V) Schizosaccharomyces pombe 972 h- M57956 atpD ParasitesGiardia lamblia WB U18938 atpD Plasmodium falciparum 3D7 L08200 atpD (V)Trypanosoma congolense IL3000 Z25814 atpD (V) Human and plants Homosapiens L09234 atpD (V) Homo sapiens M27132 atpD recA sequences BacteriaAcetobacter aceti no. 1023 S60630 recA Acetobacter altoacetigenes MH-24E05290 recA Acetobacter polyoxogenes NBI 1028 D13183 recA Acholeplasmalaidlawii 8195 M81465 recA Acidiphilium facilis ATCC 35904 D16538 recAAcidothermus cellulolyticus ATCC 43068 AJ006705 recA Acinetobactercalcoaceticus BD413/ADP1 L26100 recA Actinobacillusactinomycetemcomitans HK1651 Genome project² recA Aeromonas salmonicidaA449 U83688 recA Agrobacterium tumefaciens C58 L07902 recAAllochromatium vinosum AJ000677 recA Aquifex aeolicus VF5 AE000775 recAAquifex pyrophilus Kol5a L23135 recA Azotobacter vinelandii S96898 recABacillus stearothermophilus 10 Genome project² recA Bacillus subtilisPB1831 U87792 recA Bacillus subtilis 168 Z99112 recA Bacteroidesfragilis M63029 recA Bifidobacterium breve NCFB 2258 AF094756 recABlastochloris viridis DSM 133 AF022175 recA Bordetella pertussis 165X53457 recA Bordetella pertussis Tohama I Genome project² recA Borreliaburgdorferi Sh-2-82 U23457 recA Borrelia burgdorferi B31 AE001124 recABrevibacterium flavum MJ-233 E10390 recA Brucella abortus 2308 L00679recA Burkholderia cepacia ATCC 17616 U70431 recA Burkholderia cepaciaD90120 recA Burkholderia pseudomallei K96243 Genome project² recACampylobacter fetus subsp. fetus 23D AF020677 recA Campylobacter jejuni81-176 U03121 recA Campylobacter jejuni NCTC 11168 AL139079 recAChlamydia trachomatis L2 U16739 recA Chlamydia trachomatis D/UW-3/CXAE001335 recA Chlamydophila pneumoniae CWL029 AE001658 recA Chloroflexusaurantiacus J-10-fl AF037259 recA Clostridium acetobutylicum M94057 recAClostridium perfringens 13 U61497 recA Corynebacterium diphtheriaeNCTC13129 Genome project² recA Corynebacterium glutamicum AS019 U14965recA Corynebacterium pseudotuberculosis C231 U30387 recA Deinococcusradiodurans KD8301 AB005471 recA Deinococcus radiodurans R1 U01876 recAEnterobacter agglomerans 339 L03291 recA Enterococcus faecalis OGIXM81466 recA Erwinia carotovora X55554 recA Escherichia coli J01672 recAEscherichia coli X55552 recA Escherichia coli K-12 AE000354 recA Frankiaalni Arl3 AJ006707 recA Gluconobacter oxydans U21001 recA Haemophilusinfluenzae Rd U32687 recA Haemophilus influenzae Rd U32741 recAHaemophilus influenzae Rd L07529 recA Helicobacter pylori 69A Z35478recA Helicobacter pylori 26695 AE000536 recA Helicobacter pylori J99AE001453 recA Klebsiella pneumoniae M6H 78578 Genome project² recALactococcus lactis ML3 M88106 recA Legionella pneumophila X55453 recALeptospira biflexa serovar patoc U32625 recA Leptospira interrogansserovar pomona U29169 recA Magnetospirillum magnetotacticum MS-1 X17371recA Methylobacillus flagellatus MFK1 M35325 recA Methylomonas claraATCC 31226 X59514 recA Mycobacterium avium 104 Genome project² recAMycobacterium bovis AF122/97 Genome project² recA Mycobacterium lepraeX73822 recA Mycobacterium tuberculosis H37Rv X58485 recA Mycobacteriumtuberculosis CSU#93 Genome project² recA Mycoplasma genitalium G37U39717 recA Mycoplasma mycoides GM9 L22073 recA Mycoplasma pneumoniaeATCC 29342 MPAE000033 recA Mycoplasma pulmonis KD735 L22074 recAMyxococcus xanthus L40368 recA Myxococcus xanthus L40367 recA Neisseriaanimalis NCTC 10212 U57910 recA Neisseria cinerea LCDC 81-176 AJ223869recA Neisseria cinerea LNP 1646 U57906 recA Neisseria cinerea NCTC 10294AJ223871 recA Neisseria cinerea Vedros M601 AJ223870 recA Neisseriaelongata CCUG 2131 AJ223882 recA Neisseria elongata CCUG 4165A AJ223880recA Neisseria elongata NCTC 10660 AJ223881 recA Neisseria elongata NCTC11050 AJ223878 recA Neisseria elongata NHITCC 2376 AJ223877 recANeisseria elongata CCUG 4557 AJ223879 recA subsp. intermedia Neisseriaflava Bangor 9 AJ223873 recA Neisseria flavescens LNP 444 U57907 recANeisseria gonorrhoeae CH95 U57902 recA Neisseria gonorrhoeae FA19 X64842recA Neisseria gonorrhoeae MS11 X17374 recA Neisseria gonorrhoeae Genomeproject² recA Neisseria lactamica CCUC 7757 AJ223866 recA Neisserialactamica CCUG 7852 Y11819 recA Neisseria lactamica LCDC 77-143 Y11818recA Neisseria lactamica LCDC 80-111 AJ223864 recA Neisseria lactamicaLCDC 845 AJ223865 recA Neisseria lactamica NCTC 10617 U57905 recANeisseria lactamica NCTC 10618 AJ223863 recA Neisseria meningitidis44/46 X64849 recA Neisseria meningitidis Bangor 13 AJ223868 recANeisseria meningitidis HF116 X64848 recA Neisseria meningitidis HF130X64844 recA Neisseria meningitidis HF46 X64847 recA Neisseriameningitidis M470 X64850 recA Neisseria meningitidis N94II X64846 recANeisseria meningitidis NCTC 8249 AJ223867 recA Neisseria meningitidisP63 X64845 recA Neisseria meningitidis S3446 U57903 recA Neisseriameningitidis FAM18 Genome project² recA Neisseria mucosa LNP 405 U57908recA Neisseria mucosa Vedros M1801 AJ223875 recA Neisseria perflava CCUG17915 AJ223876 recA Neisseria perflava LCDC 85402 AJ223862 recANeisseria pharyngis var. flava NCTC 4590 U57909 recA Neisseriapolysaccharea CCUG 18031 Y11815 recA Neisseria polysaccharea CCUG 24845Y11816 recA Neisseria polysaccharea CCUG 24846 Y11814 recA Neisseriapolysaccharea INS MA 3008 Y11817 recA Neisseria polysaccharea NCTC 11858U57904 recA Neisseria sicca NRL 30016 AJ223872 recA Neisseria subflavaNRL 30017 AJ223874 recA Paracoccus denitrificans DSM 413 U59631 recAPasteurella multocida X99324 recA Porphyromonas gingivalis W83 U70054recA Prevotella ruminicola JCM 8958 U61227 recA Proteus mirabilis pG1300X14870 recA Proteus vulgaris X55555 recA Pseudomonas aeruginosa X05691recA Pseudomonas aeruginosa PAM 7 X52261 recA Pseudomonas aeruginosaPAO12 D13090 recA Pseudomonas fluorescens OE 28.3 M96558 recAPseudomonas putida L12684 recA Pseudomonas putida PpS145 U70864 recARhizobium leguminosarum VF39 X59956 recA biovar viciae Rhizobiumphaseoli CNPAF512 X62479 recA Rhodobacter capsulatus J50 X82183 recARhodobacter sphaeroides 2.4.1 X72705 recA Rhodopseudomonas palustris N 7D84467 recA Rickettsia prowazekii Madrid E AJ235273 recA Rickettsiaprowazekii Madrid E U01959 recA Serratia marcescens M22935 recA Shigellaflexneri X55553 recA Shigella sonnei KNIH104S AF101227 recASinorhizobium meliloti 2011 X59957 recA Staphylococcus aureus L25893recA Streptococcus gordonii Challis V288 L20574 recA Streptococcusmutans UA96 M81468 recA Streptococcus mutans GS-5 M61897 recAStreptococcus pneumoniae Z17307 recA Streptococcus pneumoniae R800Z34303 recA Streptococcus pyogenes NZ131 U21934 recA Streptococcuspyogenes D471 M81469 recA Streptococcus salivarius M94062 recA subsp.thermophilus Streptomyces ambofaciens DSM 40697 Z30324 recA Streptomycescoelicolor A3(2) AL020958 recA Streptomyces lividans TK24 X76076 recAStreptomyces rimosus R6 X94233 recA Streptomyces venezuelae ATCC10712U04837 recA Synechococcus sp. PR6 M29495 recA Synechocystis sp. PCC6803D90917 recA Thermotoga maritima L23425 recA Thermotoga maritima AE001823recA Thermus aquaticus L20095 recA Thermus thermophilus HB8 D17392 recAThiobacillus ferrooxidans M26933 recA Treponema denticola Genomeproject² recA Treponema pallidum Nichols AE001243 recA Vibrioanguillarum M80525 recA Vibrio cholerae 017 X71969 recA Vibrio cholerae2740-80 U10162 recA Vibrio cholerae 569B L42384 recA Vibrio choleraeM549 AF117881 recA Vibrio cholerae M553 AF117882 recA Vibrio choleraeM645 AF117883 recA Vibrio cholerae M793 AF117878 recA Vibrio choleraeM794 AF117880 recA Vibrio cholerae M967 AF117879 recA Xanthomonas citriXW47 AF006590 recA Xanthomonas oryzae AF013600 recA Xenorhabdus bovieniiT228/1 U87924 recA Xenorhabdus nematophilus AN6 AF127333 recA Yersiniapestis 231 X75336 recA Yersinia pestis CO-92 Genome project² recA Fungi,parasites, human and plants Anabaena variabilis ATCC 29413 M29680 recAArabidopsis thaliana U43652 recA (Rad51) Candida albicans U39808 recA(Dmc1) Coprinus cinereus Okayama-7 U21905 recA (Rad51) Emericellanidulans Z80341 recA (Rad51) Gallus gallus L09655 recA (Rad51) Homosapiens D13804 recA (Rad51) Homo sapiens D63882 recA (Dmc1) Leishmaniamajor Friedlin AF062379 recA (Rad51) Leishmania major Friedlin AF062380recA (Dmc1) Mus musculus D58419 recA (Dmc1) Neurospora crassa 74-OR23-1AD29638 recA (Rad51) Saccharomyces cerevisiae D10023 recA (Rad51)Schizosaccharomyces pombe Z22691 recA (Rad51) Schizosaccharomyces pombe972h- AL021817 recA (Dmc1) Tetrahymena thermophila PB9R AF064516 recA(Rad51) Trypanosoma brucei stock 427 Y13144 recA (Rad51) Ustilago maydisU62484 recA (Rad51) Xenopus laevis D38488 recA (Rad51) Xenopus laevisD38489 recA (Rad51) *tuf indicates tuf sequences, including tuf genes,fusA genes and fusA-tuf intergenic spacers. tuf (C) indicates tufsequences divergent from main (usually A and B) copies of the elongationfactor-Tu tuf (EF-1) indicates tuf sequences of the eukaryotic type(elongation factor 1α) tuf (M) indicates tuf sequences from organellar(mostly mitochondrial) origin atpD indicates atpD sequences of theF-type atpD (V) indicates atpD sequences of the V-Type recA indicatesrecA sequences recA (Rad51) indicates rad51 sequences or homologs recA(Dmc1) indicates dmc1 sequences or homologs ¹Nucleotides sequencespublished in Arch. Microbiol. 1990 153: 241-247 ²These sequences arefrom the TIGR database (http://www.tigr.org/tdb/tdb.html) ³Nucleotidessequences published in FEMS Microbiology Letters 1988 50: 101-106

TABLE 12 Bacterial species used to test the specificity of theStaphylococcus- specific amplification primers derived from tufsequences. Strain Reference number Staphylococcal species (n = 27)Staphylococcus arlettae ATCC 43957 Staphylococcus aureus ATCC 35844subsp. anaerobius Staphylococcus aureus ATCC 43300 subsp. aureusStaphylococcus auricularis ATCC 33753 Staphylococcus capitis ATCC 27840subsp. capitis Staphylococcus caprae ATCC 35538 Staphylococcus carnosusATCC 51365 Staphylococcus chromogenes ATCC 43764 Staphylococcus cohniiDSM 20260 subsp. urealyticum Staphylococcus delphini ATCC 49171Staphylococcus epidermidis ATCC 14990 Staphylococcus equorum ATCC 43958Staphylococcus felis ATCC 49168 Staphylococcus gallinarum ATCC 35539Staphylococcus haemolyticus ATCC 29970 Staphylococcus hominis ATCC 27844Staphylococcus hyicus ATCC 11249 Staphylococcus intermedius ATCC 29663Staphylococcus kloosis ATCC 43959 Staphylococcus lentus ATCC 29070Staphylococcus lugdunensis ATCC 43809 Staphylococcus saprophyticus ATCC15305 Staphylococcus schleiferi ATCC 49545 subsp. coagulansStaphylococcus sciuri ATCC 29060 subsp. sciuri Staphylococcus simulansATCC 27848 Staphylococcus warneri ATCC 27836 Staphylococcus xylosus ATCC29971 Gram-negative bacteria (n = 33) Acinetobacter baumannii ATCC 19606Bacteroides distasonis ATCC 8503 Bacteroides fragilis ATCC 25285Bulkholderia cepacia ATCC 25416 Bordetella pertussis ATCC 9797Citrobacter freundii ATCC 8090 Enterobacter aerogenes ATCC 13048Enterobacter cloacae ATCC 13047 Escherichia coli ATCC 25922 Haemophilusinfluenzae ATCC 8907 Haemophilus parahaemolyticus ATCC 10014 Haemophilusparainfluenzae ATCC 7901 Hafnia alvei ATCC 13337 Kingella indologenesATCC 25869 Klebsiella oxytoca ATCC 13182 Klebsiella pneumoniae ATCC13883 Moraxella catarrhalis ATCC 25240 Morganella morganii ATCC 25830Neisseria gonorrhoeae ATCC 35201 Neisseria meningitidis ATCC 13077Proteus mirabilis ATCC 25933 Proteus vulgaris ATCC 13315 Providenciarettgeri ATCC 9250 Providencia stuartii ATCC 29914 Pseudomonasaeruginosa ATCC 27853 Pseudomonas fluorencens ATCC 13525 Salmonellacholeraesuis ATCC 7001 Salmonella typhimurium ATCC 14028 Serratiamarcescens ATCC 8100 Shigella flexneri ATCC 12022 Shigella sonnei ATCC29930 Stenotrophomonas maltophilia ATCC 13843 Yersinia enterocoliticaATCC 9610 Other Gram-positive bacteria (n = 20) Bacillus subtilis ATCC27370 Enterococcus avium ATCC 14025 Enterococcus durans ATCC 19432Enterococcus faecalis ATCC 19433 Enterococcus faecium ATCC 19434Enterococcus flavescens ATCC 49996 Enterococcus gallinarum ATCC 49573Lactobacillus acidophilus ATCC 4356 Lactococcus lactis ATCC 11454Listeria innocua ATCC 33090 Listeria ivanovii ATCC 19119 Listeriamonocytogenes ATCC 15313 Macrococcus caseolyticus ATCC 13548Streptococcus agalactiae ATCC 13813 Streptococcus anginosus ATCC 33397Streptococcus bovis ATCC 33317 Streptococcus mutans ATCC 25175Streptococcus pneumoniae ATCC 6303 Streptococcus pyogenes ATCC 19615Streptococcus salivarius ATCC 7073

TABLE 13 Bacterial species used to test the specificity of thepenicillin-resistant Streptococcus pneumoniae assay. Strain Referencenumber Gram-positive species (n = 67) Abiotrophia adiacens ATCC 49175Abiotrophia defectiva ATCC 49176 Actinomyces pyogenes ATCC 19411Bacillus anthracis ATCC 4229 Bacillus cereus ATCC 14579 Bifidobacteriumbreve ATCC 15700 Clostridium difficile ATCC 9689 Enterococcus avium ATCC14025 Enterococcus casseliflavus ATCC 25788 Enterococcus dispar ATCC51266 Enterococcus durans ATCC 19432 Enterococcus faecalis ATCC 29212Enterococcus faecium ATCC 19434 Enterococcus flavescens ATCC 49996Enterococcus gallinarum ATCC 49573 Enterococcus hirae ATCC 8043Enterococcus mundtii ATCC 43186 Enterococcus raffinosus ATCC 49427Lactobacillus lactis ATCC 19435 Lactobacillus monocytogenes ATCC 15313Mobiluncus curtisii ATCC 35242 Peptococcus niger ATCC 27731Peptostreptococcus acones ATCC 6919 Peptostreptococcus anaerobius ATCC27337 Peptostreptococcus ATCC 2639 asaccharolyticus Peptostreptococcuslactolyticus ATCC 51172 Peptostreptococcus magnus ATCC 15794Peptostreptococcus prevotii ATCC 9321 Peptostreptococcus tetradius ATCC35098 Staphylococcus aureus ATCC 25923 Staphylococcus capitis ATCC 27840Staphylococcus epidermidis ATCC 14990 Staphylococcus haemolyticus ATCC29970 Staphylococcus hominis ATCC 27844 Staphylococcus lugdunensis ATCC43809 Staphylococcus saprophyticus ATCC 15305 Staphylococcus simulansATCC 27848 Staphylococcus. warneri ATCC 27836 Streptococcus acidominimusATCC 51726 Streptococcus agalactiae ATCC 12403 Streptococcus anginosusATCC 33397 Streptococcus bovis ATCC 33317 Streptococcus constellatusATCC 27823 Streptococcus cricetus ATCC 19624 Streptococcus cristatusATCC 51100 Streptococcus downei ATCC 33748 Streptococcus dysgalactiaeATCC 43078 Streptococcus equi ATCC 9528 Streptococcus ferus ATCC 33477Streptococcus gordonii ATCC 10558 Streptococcus intermedius ATCC 27335Streptococcus mitis ATCC 903 Streptococcus mitis LSPQ 2583 Streptococcusmitis ATCC 49456 Streptococcus mutans ATCC 27175 Streptococcus oralisATCC 10557 Streptococcus oralis ATCC 9811 Streptococcus oralis ATCC35037 Streptococcus parasanguinis ATCC 15912 Streptococcus parauberisATCC 6631 Streptococcus rattus ATCC 15912 Streptococcus salivarius ATCC7073 Streptococcus sanguinis ATCC 10556 Streptococcus suis ATCC 43765Streptococcus uberis ATCC 19436 Streptococcus vestibularis ATCC 49124Gram-negative species (n = 33) Actinetobacter baumannii ATCC 19606Bordetella pertussis ATCC 9797 Citrobacter diversus ATCC 27028Citrobacter freundii ATCC 8090 Enterobacter aerogenes ATCC 13048Enterobacter agglomerans ATCC 27155 Enterobacter cloacae ATCC 13047Escherichia coli ATCC 25922 Haemophilus ducreyi ATCC 33940 Haemophilushaemolyticus ATCC 33390 Haemophilus influenzae ATCC 9007 Haemophilusparainfluenzae ATCC 7901 Hafnia alvei ATCC 13337 Klebsiella oxytoca ATCC13182 Klebsiella pneumoniae ATCC 13883 Moraxella atlantae ATCC 29525Moraxella catarrhalis ATCC 43628 Moraxella morganii ATCC 13077 Neisseriagonorrhoeae ATCC 35201 Neisseria meningitidis ATCC 13077 Proteusmirabilis ATCC 25933 Proteus vulgaris ATCC 13315 Providenciaalcalifaciens ATCC 9886 Providencia rettgeri ATCC 9250 Providenciarustigianii ATCC 33673 Providencia stuartii ATCC 33672 Pseudomonasaeruginosa ATCC 35554 Pseudomonas fluorescens ATCC 13525 Pseudomonasstutzeri ATCC 17588 Salmonella typhimurium ATCC 14028 Serratiamarcescens ATCC 13880 Shigella flexneri ATCC 12022 Yersinaenterocolitica ATCC 9610

TABLE 14 Bacterial species (n = 104) detected by the plateletcontaminants assay. Bold characters indicate the major bacterialcontaminants found in platelet concentrates. Abiotrophia adiacensAbiotrophia defectiva Acinetobacter baumannii Acinetobacter lwoffiAerococcus viridans Bacillus anthracis

Brucella abortus Burkholderia cepacia Citrobacter diversus Citrobacterfreundii Enterobacter aerogenes Enterobacter agglomerans

Enterococcus avium Enterococcus casseliflavus Enterococcus disparEnterococcus durans Enterococcus faecalis Enterococcus faeciumEnterococcus flavescens Enterococcus gallinarum Enterococcus mundtiiEnterococcus raffinosus Enterococcus solitarius

Gemella morbillorum Haemophilus ducreyi Haemophilus haemolyticusHaemophilus influenzae Haemophilus parahaemolyticus Haemophilusparainfluenzae Hafnia alvei Kingella kingae

Legionella pneumophila Megamonas hypermegale Moraxella atlantaeMoraxella catarrhalis Morganella morganii Neisseria gonorrheae Neisseriameningitidis Pasteurella aerogenes Pasteurella multocidaPeptostreptococcus magnus Proteus mirabilis Providencia alcalifaciensProvidencia rettgeri Providencia rustigianii Providencia stuartii

Pseudomonas fluorescens Pseudomonas stutzeri Salmonella bongori

Salmonella enteritidis Salmonella gallinarum Salmonella typhimuriumSerratia liquefaciens

Shigella flexneri Shigella sonnei

Staphylococcus capitis

Staphylococcus haemolyticus Staphylococcus hominis Staphylococcuslugdunensis Staphylococcus saprophyticus Staphylococcus simulansStaphylococcus warneri Stenotrophomonas maltophilia Streptococcusacidominimus

Streptococcus anginosus Streptococcus bovis Streptococcus constellatusStreptococcus cricetus Streptococcus cristatus Streptococcusdysgalactiae Streptococcus equi Streptococcus ferus Streptococcusgordonii Streptococcus intermedius Streptococcus macacae Streptococcusmitis

Streptococcus oralis Streptococcus parasanguinis Streptococcusparauberis Streptococcus pneumoniae

Streptococcus ratti

Streptococcus sanguinis Streptococcus sobrinus Streptococcus uberisStreptococcus vestibularis Vibrio cholerae Yersinia enterocoliticaYersinia pestis

TABLE 15 Microorganisms identified by commercial systems¹. Abiotrophiaadiacens (Streptococcus adjacens) Abiotrophia defectiva (Streptococcusdefectivus) Achromobacter species Acidaminococcus fermentansAcinetobacter alcaligenes Acinetobacter anitratus Acinetobacterbaumannii Acinetobacter calcoaceticus Acinetobacter calcoaceticus biovaranitratus Acinetobacter calcoaceticus biovar lwoffi Acinetobactergenomospecies Acinetobacter haemolyticus Acinetobacter johnsoniiAcinetobacter junii Acinetobacter lwoffii Acinetobacter radioresistensAcinetobacter species Actinobacillus actinomycetemcomitansActinobacillus capsulatus Actinobacillus equuli Actinobacillus hominisActinobacillus lignieresii Actinobacillus pleuropneumoniaeActinobacillus species Actinobacillus suis Actinobacillus ureaeActinomyces bovis Actinomyces israelii Actinomyces meyeri Actinomycesnaeslundii Actinomyces neuii subsp. anitratus Actinomyces neuii subsp.neuii Actinomyces odontolyticus Actinomyces pyogenes Actinomycesradingae Actinomyces species Actinomyces turicensis Actinomyces viscosusAerococcus species Aerococcus viridans Aeromonas caviae Aeromonashydrophila Aeromonas hydrophila group Aeromonas jandaei Aeromonassalmonicida Aeromonas salmonicida subsp. achromogenes Aeromonassalmonicida subsp. masoucida Aeromonas salmonicida subsp. salmonicidaAeromonas schubertii Aeromonas sobria Aeromonas species Aeromonas trotaAeromonas veronii Aeromonas veronii biovar sobria Aeromonas veroniibiovar veronii Agrobacterium radiobacter Agrobacterium speciesAgrobacterium tumefaciens Alcaligenes denitrificans Alcaligenes faecalisAlcaligenes odorans Alcaligenes odorans (Alcaligenes faecalis)Alcaligenes species Alcaligenes xylosoxidans Alcaligenes xylosoxidanssubsp. denitrificans Alcaligenes xylosoxidans subsp. xylosoxidansAlloiococcus otitis Anaerobiospirillum succiniciproducens Anaerovibriolipolytica Arachnia propionica Arcanobacterium (Actinomyces) bernardiaeArcanobacterium (Actinomyces) pyogenes Arcanobacterium haemolyticumArcobacter cryaerophilus (Campylobacter cryaerophila) Arthrobacterglobiformis Arthrobacter species Arxiozyma telluris (Torulopsispintolopesii) Atopobium minutum (Lactobacillus minutus) Aureobacteriumspecies Bacillus amyloliquefaciens Bacillus anthracis Bacillus badiusBacillus cereus Bacillus circulans Bacillus coagulans Bacillus firmusBacillus lentus Bacillus licheniformis Bacillus megaterium Bacillusmycoides Bacillus pantothenticus Bacillus pumilus Bacillus speciesBacillus sphaericus Bacillus stearothermophilus Bacillus subtilisBacillus thuringiensis Bacteroides caccae Bacteroides capillosusBacteroides distasonis Bacteroides eggerthii Bacteroides fragilisBacteroides merdae Bacteroides ovatus Bacteroides species Bacteroidessplanchnicus Bacteroides stercoris Bacteroides thetaiotaomicronBacteroides uniformis Bacteroides ureolyticus (B. corrodens) Bacteroidesvulgatus Bergeyella (Weeksella) zoohelcum Bifidobacterium adolescentisBifidobacterium bifidum Bifidobacterium breve Bifidobacterium dentiumBifidobacterium infantis Bifidobacterium species Blastoschizomyces(Dipodascus) capitatus Bordetella avium Bordetella bronchisepticaBordetella parapertussis Bordetella pertussis Bordetella speciesBorrelia species Branhamella (Moraxella) catarrhalis Branhamella speciesBrevibacillus brevis Brevibacillus laterosporus Brevibacterium caseiBrevibacterium epidermidis Brevibacterium linens Brevibacterium speciesBrevundimonas (Pseudomonas) diminuta Brevundimonas (Pseudomonas)vesicularis Brevundimonas species Brochothrix thermosphacta Brucellaabortus Brucella canis Brucella melitensis Brucella ovis Brucellaspecies Brucella suis Budvicia aquatica Burkholderia (Pseudomonas)cepacia Burkholderia (Pseudomonas) gladioli Burkholderia (Pseudomonas)mallei Burkholderia (Pseudomonas) pseudomallei Burkholderia speciesButtiauxella agrestis Campylobacter coli Campylobacter concisusCampylobacter fetus Campylobacter fetus subsp. fetus Campylobacter fetussubsp. venerealis Campylobacter hyointestinalis Campylobacter jejunisubsp. doylei Campylobacter jejuni subsp. jejuni Campylobacter lariCampylobacter lari subsp. UPTC Campylobacter mucosalis Campylobacterspecies Campylobacter sputorum Campylobacter sputorum subsp. bubulusCampylobacter sputorum subsp. fecalis Campylobacter sputorum subsp.sputorum Campylobacter upsaliensis Candida (Clavispora) lusitaniaeCandida (Pichia) guilliermondii Candida (Torulopsis) glabrata Candidaalbicans Candida boidinii Candida catenulata Candida ciferrii Candidacolliculosa Candida conglobata Candida curvata (Cryptococcus curvatus)Candida dattila Candida dubliniensis Candida famata Candida globosaCandida hellenica Candida holmii Candida humicola Candida inconspicuaCandida intermedia Candida kefyr Candida krusei Candida lambica Candidamagnoliae Candida maris Candida melibiosica Candida membranaefaciensCandida norvegensis Candida norvegica Candida parapsilosis Candidaparatropicalis Candida pelliculosa Candida pseudotropicalis Candidapulcherrima Candida ravautii Candida rugosa Candida sake Candidasilvicola Candida species Candida sphaerica Candida stellatoidea Candidatenuis Candida tropicalis Candida utilis Candida valida Candida viniCandida viswanathii Candida zeylanoides Capnocytophaga gingivalisCapnocytophaga ochracea Capnocytophaga species Capnocytophaga sputigenaCardiobacterium hominis Carnobacterium divergens Carnobacteriumpiscicola CDC group ED-2 CDC group EF4 (Pasteurella sp.) CDC group EF-4ACDC group EF-4B CDC group EQ-Z CDC group HB-5 CDC group II K-2 CDC groupIV C-2 (Bordetella-like) CDC group M5 CDC group M6 Cedecea davisaeCedecea lapagei Cedecea neteri Cedecea species Cellulomonas (Oerskovia)turbata Cellulomonas species Chlamydia species Chromobacterium violaceumChryseobacterium (Flavobacterium) indologenes Chryseobacterium(Flavobacterium) meningosepticum Chryseobacterium gleum Chryseobacteriumspecies Chryseomonas indologenes Citeromyces matritensis Citrobacteramalonaticus Citrobacter braakii Citrobacter diversus Citrobacterfarmeri Citrobacter freundii Citrobacter freundii complex Citrobacterkoseri Citrobacter sedlakii Citrobacter species Citrobacter werkmaniiCitrobacter youngae Clostridium acetobutylicum Clostridium baratiClostridium beijerinckii Clostridium bifermentans Clostridium botulinumClostridium botulinum (NP) B&F Clostridium botulinum (NP) E Clostridiumbotulinum (P) A&H Clostridium botulinum (P) F Clostridium botulinum G1Clostridium botulinum G2 Clostridium butyricum Clostridium cadaverisClostridium chauvoei Clostridium clostridiiforme Clostridium difficileClostridium fallax Clostridium glycolicum Clostridium hastiformeClostridium histolyticum Clostridium innocuum Clostridium limosumClostridium novyi Clostridium novyi A Clostridium paraputrificumClostridium perfringens Clostridium putrificum Clostridium ramosumClostridium septicum Clostridium sordellii Clostridium speciesClostridium sphenoides Clostridium sporogenes Clostridium subterminaleClostridium tertium Clostridium tetani Clostridium tyrobutyricumComamonas (Pseudomonas) acidovorans Comamonas (Pseudomonas) testosteroniComamonas species Corynebacterium accolens Corynebacterium afermentansCorynebacterium amycolatum Corynebacterium aquaticum Corynebacteriumargentoratense Corynebacterium auris Corynebacterium bovisCorynebacterium coyleae Corynebacterium cystitidis Corynebacteriumdiphtheriae Corynebacterium diphtheriae biotype belfanti Corynebacteriumdiphtheriae biotype gravis Corynebacterium diphtheriae biotypeintermedius Corynebacterium diphtheriae biotype mitis Corynebacteriumflavescens Corynebacterium glucuronolyticum Corynebacteriumglucuronolyticum- seminale Corynebacterium group A Corynebacterium groupA-4 Corynebacterium group A-5 Corynebacterium group ANF Corynebacteriumgroup B Corynebacterium group B-3 Corynebacterium group FCorynebacterium group F-1 Corynebacterium group F-2 Corynebacteriumgroup G Corynebacterium group G-1 Corynebacterium group G-2Corynebacterium group I Corynebacterium group I-2 Corynebacteriumjeikeium (group JK) Corynebacterium kutscheri (C. murium)Corynebacterium macginleyi Corynebacterium minutissimum Corynebacteriumpilosum Corynebacterium propinquum Corynebacterium pseudodiphtheriticumCorynebacterium pseudotuberculosis Corynebacterium pyogenesCorynebacterium renale Corynebacterium renale group Corynebacteriumseminale Corynebacterium species Corynebacterium striatum (C. flavidum)Corynebacterium ulcerans Corynebacterium urealyticum (group D2)Corynebacterium xerosis Cryptococcus albidus Cryptococcus aterCryptococcus cereanus Cryptococcus gastricus Cryptococcus humicolusCryptococcus lactativorus Cryptococcus laurentii Cryptococcus luteolusCryptococcus melibiosum Cryptococcus neoformans Cryptococcus speciesCryptococcus terreus Cryptococcus uniguttulatus Debaryomyces hanseniiDebaryomyces marama Debaryomyces polymorphus Debaryomyces speciesDermabacter hominis Dermacoccus (Micrococcus) nishinomiyaensis Dietziaspecies Edwardsiella hoshinae Edwardsiella ictaluri Edwardsiella speciesEdwardsiella tarda Eikenella corrodens Empedobacter brevis(Flavobacterium breve) Enterobacter aerogenes Enterobacter agglomeransEnterobacter amnigenus Enterobacter amnigenus asburiae (CDC entericgroup 17) Enterobacter amnigenus biogroup 1 Enterobacter amnigenusbiogroup 2 Enterobacter asburiae Enterobacter cancerogenus Enterobactercloacae Enterobacter gergoviae Enterobacter hormaechei Enterobacterintermedius Enterobacter sakazakii Enterobacter species Enterobactertaylorae Enterobacter taylorae (CDC enteric group 19) Enterococcus(Streptococcus) cecorum Enterococcus (Streptococcus) faecalis (Group D)Enterococcus (Streptococcus) faecium (Group D) Enterococcus(Streptococcus) saccharolyticus Enterococcus avium (Group D)Enterococcus casseliflavus (Steptococcus faecium subsp. casseliflavus)Enterococcus durans (Streptococcus faecium subsp. durans) (Group D)Enterococcus gallinarum Enterococcus hirae Enterococcus malodoratusEnterococcus mundtii Enterococcus raffinosus Enterococcus speciesErwinia amylovora Erwinia carotovora Erwinia carotovora subsp.atroseptica Erwinia carotovora subsp. betavasculorum Erwinia carotovorasubsp. carotovora Erwinia chrysanthemi Erwinia cypripedii Erwiniamallotivora Erwinia nigrifluens Erwinia quercina Erwinia rhaponticiErwinia rubrifaciens Erwinia salicis Erwinia species Erysipelothrixrhusiopathiae Erysipelothrix species Escherichia blattae Escherichiacoli Escherichia coli A-D Escherichia coli O157:H7 Escherichiafergusonii Escherichia hermannii Escherichia species Escherichiavulneris Eubacterium aerofaciens Eubacterium alactolyticum Eubacteriumlentum Eubacterium limosum Eubacterium species Ewingella americanaFilobasidiella neoformans Filobasidium floriforme Filobasidiumuniguttulatum Flavimonas oryzihabitans Flavobacterium gleumFlavobacterium indologenes Flavobacterium odoratum Flavobacteriumspecies Francisella novicida Francisella philomiragia Francisellaspecies Francisella tularensis Fusobacterium mortiferum Fusobacteriumnecrogenes Fusobacterium necrophorum Fusobacterium nucleatumFusobacterium species Fusobacterium varium Gaffkya species Gardnerellavaginalis Gemella haemolysans Gemella morbillorum Gemella speciesGeotrichum candidum Geotrichum fermentans Geotrichum penicillarumGeotrichum penicillatum Geotrichum species Gordona species Haemophilusaegyptius Haemophilus aphrophilus Haemophilus ducreyi Haemophilushaemoglobinophilus Haemophilus haemolyticus Haemophilus influenzaeHaemophilus influenzae biotype I Haemophilus influenzae biotype IIHaemophilus influenzae biotype III Haemophilus influenzae biotype IVHaemophilus influenzae biotype V Haemophilus influenzae biotype VIHaemophilus influenzae biotype VII Haemophilus influenzae biotype VIIIHaemophilus paragallinarum Haemophilus parahaemolyticus Haemophilusparainfluenzae Haemophilus parainfluenzae biotype I Haemophilusparainfluenzae biotype II Haemophilus parainfluenzae biotype IIIHaemophilus parainfluenzae biotype IV Haemophilus parainfluenzae biotypeV Haemophilus parainfluenzae biotype VI Haemophilus parainfluenzaebiotype VII Haemophilus parainfluenzae biotype VIII Haemophilusparaphrohaemolyticus Haemophilus paraphrophilus Haemophilus segnisHaemophilus somnus Haemophilus species Hafnia alvei Hanseniasporaguilliermondii Hanseniaspora uvarum Hanseniaspora valbyensis Hansenulaanomala Hansenula holstii Hansenula polymorpha Helicobacter(Campylobacter) cinaedi Helicobacter (Campylobacter) fennelliaeHelicobacter (Campylobacter) pylori Issatchenkia orientalis Kingelladenitrificans Kingella indologenes Kingella kingae Kingella speciesKlebsiella ornithinolytica Klebsiella oxytoca Klebsiella planticolaKlebsiella pneumoniae subsp. ozaenae Klebsiella pneumoniae subsp.pneumoniae Klebsiella pneumoniae subsp. rhinoscleromatis Klebsiellaspecies Klebsiella terrigena Kloeckera apiculata Kloeckera apisKloeckera japonica Kloeckera species Kluyvera ascorbata Kluyveracryocrescens Kluyvera species Kluyveromyces lactis Kluyveromycesmarxianus Kluyveromyces thermotolerans Kocuria (Micrococcus) kristinaeKocuria (Micrococcus) rosea Kocuria (Micrococcus) varians Koserellatrabulsii Kytococcus (Micrococcus) sedentarius Lactobacillus (Weissella)viridescens Lactobacillus A Lactobacillus acidophilus Lactobacillus BLactobacillus brevis Lactobacillus buchneri Lactobacillus caseiLactobacillus casei subsp. casei Lactobacillus casei subsp. lactosusLactobacillus casei subsp. rhamnosus Lactobacillus catenaformisLactobacillus cellobiosus Lactobacillus collinoides Lactobacilluscoprophilus Lactobacillus crispatus Lactobacillus curvatus Lactobacillusdelbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp.delbrueckii Lactobacillus delbrueckii subsp. lactis Lactobacillusfermentum Lactobacillus fructivorans Lactobacillus helveticusLactobacillus helveticus subsp. jugurti Lactobacillus jenseniiLactobacillus lindneri Lactobacillus minutus Lactobacillus paracaseisubsp. paracasei Lactobacillus pentosus Lactobacillus plantarumLactobacillus salivarius Lactobacillus salivarius var. saliciniusLactobacillus species Lactococcus diacitilactis Lactococcus garvieaeLactococcus lactis subsp. cremoris Lactococcus lactis subsp.diacitilactis Lactococcus lactis subsp. hordniae Lactococcus lactissubsp. lactis Lactococcus plantarum Lactococcus raffinolactis Leclerciaadecarboxylata Legionella species Leminorella species Leptospira speciesLeptotrichia buccalis Leuconostoc (Weissella) paramesenteroidesLeuconostoc carnosum Leuconostoc citreum Leuconostoc gelidum Leuconostoclactis Leuconostoc mesenteroides Leuconostoc mesenteroides subsp.cremoris Leuconostoc mesenteroides subsp. dextranicum Leuconostocmesenteroides subsp. mesenteroides Leuconostoc species Listeria grayiListeria innocua Listeria ivanovii Listeria monocytogenes Listeriamurrayi Listeria seeligeri Listeria species Listeria welshimeriMegasphaera elsdenii Methylobacterium mesophilicum Metschnikowiapulcherrima Microbacterium species Micrococcus luteus Micrococcus lylaeMicrococcus species Mobiluncus curtisii Mobiluncus mulieris Mobiluncusspecies Moellerella wisconsensis Moraxella (Branhamella) catarrhalisMoraxella atlantae Moraxella bovis Moraxella lacunata Moraxellanonliquefaciens Moraxella osloensis Moraxella phenylpyruvica Moraxellaspecies Morganella morganii Morganella morganii subsp. morganiiMorganella morganii subsp. sibonii Mycobacterium africanum Mycobacteriumasiaticum Mycobacterium avium Mycobacterium bovis Mycobacterium chelonaeMycobacterium fortuitum Mycobacterium gordonae Mycobacterium kansasiiMycobacterium malmoense Mycobacterium marinum Mycobacterium phleiMycobacterium scrofulaceum Mycobacterium smegmatis Mycobacterium speciesMycobacterium tuberculosis Mycobacterium ulcerans Mycobacterium xenopiMycoplasma fermentans Mycoplasma hominis Mycoplasma orale Mycoplasmapneumoniae Mycoplasma species Myroides species Neisseria cinereaNeisseria elongata subsp. elongata Neisseria flava Neisseria flavescensNeisseria gonorrhoeae Neisseria lactamica Neisseria meningitidisNeisseria mucosa Neisseria perflava Neisseria polysaccharea Neisseriasaprophytes Neisseria sicca Neisseria subflava Neisseria weaveriNeisseria weaveri (CDC group M5) Nocardia species Ochrobactrum anthropiOerskovia species Oerskovia xanthineolytica Oligella (Moraxella)urethralis Oligella species Oligella ureolytica Paenibacillus alveiPaenibacillus macerans Paenibacillus polymyxa Pantoea agglomeransPantoea ananas (Erwinia uredovora) Pantoea dispersa Pantoea speciesPantoea stewartii Pasteurella (Haemophilus) avium Pasteurella aerogenesPasteurella gallinarum Pasteurella haemolytica Pasteurella haemolyticusPasteurella multocida Pasteurella multocida SF Pasteurella multocidasubsp. multocida Pasteurella multocida subsp. septica Pasteurellapneumotropica Pasteurella species Pasteurella ureae Pediococcusacidilactici Pediococcus damnosus Pediococcus pentosaceus Pediococcusspecies Peptococcus niger Peptococcus species Peptostreptococcusanaerobius Peptostreptococcus asaccharolyticus Peptostreptococcusindolicus Peptostreptococcus magnus Peptostreptococcus microsPeptostreptococcus parvulus Peptostreptococcus prevotiiPeptostreptococcus productus Peptostreptococcus speciesPeptostreptococcus tetradius Phaecoccomyces exophialiae Photobacteriumdamselae Pichia (Hansenula) anomala Pichia (Hansenula) jadinii Pichia(Hansenula) petersonii Pichia angusta (Hansenula polymorpha) Pichiacarsonii (P. vini) Pichia etchellsii Pichia farinosa Pichia fermentansPichia membranaefaciens Pichia norvegensis Pichia ohmeri Pichiaspartinae Pichia species Plesiomonas shigelloides Porphyromonasasaccharolytica Porphyromonas endodontalis Porphyromonas gingivalisPorphyromonas levii Prevotella (Bacteroides) buccae Prevotella(Bacteroides) buccalis Prevotella (Bacteroides) corporis Prevotella(Bacteroides) denticola Prevotella (Bacteroides) loescheii Prevotella(Bacteroides) oralis Prevotella (Bacteroides) disiens Prevotella(Bacteroides) oris Prevotella bivia (Bacteroides bivius) Prevotellaintermedia (Bacteroides intermedius) Prevotella melaninogenica(Bacteroides melaninogenicus) Prevotella ruminicola Propionibacteriumacnes Propionibacterium avidum Propionibacterium granulosumPropionibacterium propionicum Propionibacterium species Proteusmirabilis Proteus penneri Proteus species Proteus vulgaris Protothecaspecies Prototheca wickerhamii Prototheca zopfii Providenciaalcalifaciens Providencia heimbachae Providencia rettgeri Providenciarustigianii Providencia species Providencia stuartii Providenciastuartii urea+ Pseudomonas (Chryseomonas) luteola Pseudomonasacidovorans Pseudomonas aeruginosa Pseudomonas alcaligenes Pseudomonascepacia Pseudomonas chlororaphis (P. aureofaciens) Pseudomonasfluorescens Pseudomonas fluorescens group Pseudomonas mendocinaPseudomonas pseudoalcaligenes Pseudomonas putida Pseudomonas speciesPseudomonas stutzeri Pseudomonas testosteroni Pseudomonas vesicularisPseudoramibacter (Eubacterium) alactolyticus Psychrobacter (Moraxella)phenylpyruvicus Rahnella aquatilis Ralstonia (Pseudomonas, Burkholderia)pickettii Rhodococcus (Corynebacterium) equi Rhodococcus speciesRhodosporidium toruloides Rhodotorula glutinis Rhodotorula minutaRhodotorula mucilaginosa (R. rubra) Rhodotorula species Rickettsiaspecies Rothia dentocariosa Saccharomyces cerevisiae Saccharomycesexiguus Saccharomyces kluyverii Saccharomyces species Sakaguchiadacryoides (Rhodosporidium dacryoidum) Salmonella arizonae Salmonellacholeraesuis Salmonella enteritidis Salmonella gallinarum Salmonellaparatyphi A Salmonella paratyphi B Salmonella pullorum Salmonellaspecies Salmonella typhi Salmonella typhimurium Salmonella typhisuisSalmonella/Arizona Serratia ficaria Serratia fonticola Serratia grimesiiSerratia liquefaciens Serratia marcescens Serratia odorifera Serratiaodorifera type 1 Serratia odorifera type 2 Serratia plymuthica Serratiaproteamaculans Serratia proteamaculans subsp. proteamaculans Serratiaproteamaculans subsp. quinovora Serratia rubidaea Serratia speciesShewanella (Pseudomonas, Alteromonas) putrefaciens Shigella boydiiShigella dysenteriae Shigella flexneri Shigella sonnei Shigella speciesSphingobacterium multivorum Sphingobacterium species Sphingobacteriumspiritivorum Sphingobacterium thalpophilum Sphingomonas (Pseudomonas)paucimobilis Sporidiobolus salmonicolor Sporobolomyces roseusSporobolomyces salmonicolor Sporobolomyces species Staphylococcus(Peptococcus) saccharolyticus Staphylococcus arlettae Staphylococcusaureus Staphylococcus aureus (Coagulase- negative) Staphylococcusauricularis Staphylococcus capitis Staphylococcus capitis subsp. capitisStaphylococcus capitis subsp. ureolyticus Staphylococcus capraeStaphylococcus carnosus Staphylococcus caseolyticus Staphylococcuschromogenes Staphylococcus cohnii Staphylococcus cohnii subsp. cohniiStaphylococcus cohnii subsp. urealyticum Staphylococcus epidermidisStaphylococcus equorum Staphylococcus gallinarum Staphylococcushaemolyticus Staphylococcus hominis Staphylococcus hominis subsp.hominis Staphylococcus hominis subsp. novobiosepticus Staphylococcushyicus Staphylococcus intermedius Staphylococcus kloosii Staphylococcuslentus Staphylococcus lugdunensis Staphylococcus saprophyticusStaphylococcus schleiferi Staphylococcus sciuri Staphylococcus simulansStaphylococcus species Staphylococcus warneri Staphylococcus xylosusStenotrophomonas (Xanthomonas) maltophilia Stephanoascus ciferriiStomatococcus mucilaginosus Streptococcus acidominimus Streptococcusagalactiae Streptococcus agalactiae (Group B) Streptococcus agalactiaehemolytic Streptococcus agalactiae non- hemolytic Streptococcusalactolyticus Streptococcus anginosus Streptococcus anginosus (Group D,nonenterococci) Streptococcus beta-hemolytic group A Streptococcusbeta-hemolytic non- group A or B Streptococcus beta-hemolytic non- groupA Streptococcus beta-hemolytic Streptococcus bovis (Group D,nonenterococci) Streptococcus bovis I Streptococcus bovis IIStreptococcus canis Streptococcus constellatus Streptococcusconstellatus (Streptococcus milleri I) Streptococcus constellatus(viridans Streptococcus) Streptococcus downei Streptococcus dysgalactiaesubsp. dysgalactiae Streptococcus dysgalactiae subsp. equisimilisStreptococcus equi (Group C/Group G Streptococcus) Streptococcus equisubsp. equi Streptococcus equi subsp. zooepidemicus Streptococcusequinus Streptococcus equinus (Group D, nonenterococci) Streptococcusequisimilis Streptococcus equisimulis (Group C/Group G Streptococcus)Streptococcus Gamma (non)-hemolytic Streptococcus gordonii StreptococcusGroup B Streptococcus Group C Streptococcus Group D Streptococcus GroupE Streptococcus Group F Streptococcus Group G Streptococcus Group LStreptococcus Group P Streptococcus Group U Streptococcus intermediusStreptococcus intermedius (Streptococcus milleri II) Streptococcusintermedius (viridans Streptococcus) Streptococcus milleri groupStreptococcus mitis Streptococcus mitis (viridans Streptococcus)Streptococcus mitis group Streptococcus mutans Streptococcus mutans(viridans Streptococcus) Streptococcus oralis Streptococcus parasanguisStreptococcus pneumoniae Streptococcus porcinus Streptococcus pyogenesStreptococcus pyogenes (Group A) Streptococcus salivarius Streptococcussalivarius (viridans Streptococcus) Streptococcus salivarius subsp.salivarius Streptococcus salivarius subsp. thermophilus Streptococcussanguis Streptococcus sanguis I (viridans Streptococcus) Streptococcussanguis II Streptococcus sanguis II (viridans Streptococcus)Streptococcus sobrinus Streptococcus species Streptococcus suis IStreptococcus suis II Streptococcus uberis Streptococcus uberis(viridans Streptococcus) Streptococcus vestibularis Streptococcuszooepidemicus Streptococcus zooepidemicus (Group C) Streptomycessomaliensis Streptomyces species Suttonella (Kingella) indologenesTatumella ptyseos Tetragenococcus (Pediococcus) halophilus Torulasporadelbrueckii (Saccharomyces rosei) Torulopsis candida Torulopsishaemulonii Torulopsis inconspicua Treponema species Trichosporon asahiiTrichosporon asteroides Trichosporon beigelii Trichosporon cutaneumTrichosporon inkin Trichosporon mucoides Trichosporon ovoidesTrichosporon pullulans Trichosporon species Turicella otitidisUreaplasma species Ureaplasma urealyticum Veillonella parvula (V.alcalescens) Veillonella species Vibrio alginolyticus Vibrio choleraeVibrio damsela Vibrio fluvialis Vibrio furnissii Vibrio harveyi Vibriohollisae Vibrio metschnikovii Vibrio mimicus Vibrio parahaemolyticusVibrio species Vibrio species SF Vibrio vulnificus Weeksella (Bergeylla)virosa Weeksella species Weeksella virosa Williopsis (Hansenula)saturnus Xanthomonas campestris Xanthomonas species Yarrowia (Candida)lipolytica Yersinia aldovae Yersinia enterocolitica Yersiniaenterocolitica group Yersinia frederiksenii Yersinia intermedia Yersiniaintermedius Yersinia kristensenii Yersinia pestis Yersiniapseudotuberculosis Yersinia pseudotuberculosis SF Yersinia ruckeriYersinia species Yokenella regensburgei Yokenella regensburgei(Koserella trabulsii) Zygoascus hellenicus Zygosaccharomyces species¹The list includes microorganisms that may be identified by APIidentification test systems and VITEK ® automated identification systemfrom bioMérieux Inc., or by the MicroScan ®-WalkAway ® automated systemsfrom Dade Behring. Identification relies on classical identificationmethods using batteries of biochemical and other phenotypical tests.

TABLE 16 tuf gene sequences obtained in our laboratory (Example 42).GenBank Species Strain no. Gene  Accession no.* Abiotrophia adiacensATCC49175 tuf AF124224 Enterococcus avium ATCC14025 tufA AF124220 tufBAF274715 Enterococcus casseliflavus ATCC25788 tufA AF274716 tufBAF274717 Enterococcus cecorum ATCC43198 tuf AF274718 Enterococcuscolumbae ATCC51263 tuf AF274719 Enterococcus dispar ATCC51266 tufAAF274720 tufB AF274721 Enterococcus durans ATCC19432 tufA AF274722 tufBAF274723 Enterococcus faecalis ATCC29212 tuf AF124221 Enterococcusfaecium ATCC 19434 tufA AF124222 tufB AF274724 Enterococcus gallinarumATCC49573 tufA AF124223 tufB AF274725 Enterococcus hirae ATCC8043 tufAAF274726 tufB AF274727 Enterococcus malodoratus ATCC43197 tufA AF274728tufB AF274729 Enterococcus mundtii ATCC43186 tufA AF274730 tufB AF274731Enterococcus pseudoavium ATCC49372 tufA AF274732 tufB AF274733Enterococcus raffinosus ATCC49427 tufA AF274734 tufB AF274735Enterococcus saccharolyticus ATCC43076 tuf AF274736 Enterococcussolitarius ATCC49428 tuf AF274737 Enterococcus sulfureus ATCC49903 tufAF274738 Lactococcus lactis ATCC11154 tuf AF274745 Listeriamonocytogenes ATCC15313 tuf AF274746 Listeria seeligeri ATCC35967 tufAF274747 Staphylococcus aureus ATCC25923 tuf AF274739 Staphylococcusepidermidis ATCC14990 tuf AF274740 Streptococcus mutans ATCC25175 tufAF274741 Streptococcus pneumoniae ATCC6303 tuf AF274742 Streptococcuspyogenes ATCC19615 tuf AF274743 Streptococcus suis ATCC43765 tufAF274744 *Corresponding sequence ID NO. for the above ATCC strains aregiven in table 7.

TABLE 17 tuf gene sequences selected from databases for Example 42.Species Gene Accession no.* Agrobacterium tumefaciens tufA X99673 tufBX99674 Anacystis nidulans tuf X17442 Aquifex aeolicus tufA AE000657 tufBAE000657 Bacillus stearothermophilus tuf AJ000260 Bacillus subtilis tufAL009126 Bacteroides fragilis tuf P33165 Borrelia burgdorferi tufAE000783 Brevibacterium linens tuf X76863 Bulkholderia cepacia tufP33167 Campylobacter jejuni tufB Y17167 Chlamydia pneumoniae tufAE001363 Chlamydia trachomatis tuf M74221 Corynebacterium glutamicum tufX77034 Cytophaga lytica tuf X77035 Deinococcus radiodurans tuf AE000513Escherichia coli tufA J01690 tufB J01717 Fervidobacterium islandicum tufY15788 Haemophilus influenzae tufA L42023 tufB L42023 Helicobacterpylori tuf AE000511 Homo sapiens (Human) EF-1α X03558 Methanococcusjannaschii EF-1α U67486 Mycobacterium leprae tuf D13869 Mycobacteriumtuberculosis tuf X63539 Mycoplasma genitalium tuf L43967 Mycoplasmapneumoniae tuf U00089 Neisseria gonorrhoeae tufA L36380 Nicotianatabacum (Tobacco) EF-1α U04632 Peptococcus niger tuf X76869 Planobisporarosea tuf1 U67308 Saccharomyces cerevisiae (Yeast) EF-1α X00779Salmonella typhimurium tufA X55116 tufB X55117 Shewanella putrefacienstuf P33169 Spirochaeta aurantia tuf X76874 Spirulina platensis tufAX15646 Streptomyces aureofaciens tuf1 AF007125 Streptomyces cinnamoneustuf1 X98831 Streptomyces coelicolor tuf1 X77039 tuf3 X77040 Streptomycescollinus tuf1 S79408 Streptomyces ramocissimus tuf1 X67057 tuf2 X67058tuf3 X67059 Synechocystis sp. tuf AB001339 Taxeobacter ocellatus tufX77036 Thermotoga maritima tuf AE000512 Thermus aquaticus tuf X66322Thermus thermophilus tuf X06657 Thiobacillus cuprinus tuf U78300Treponema pallidum tuf AE000520 Wolinella succinogenes tuf X76872*Sequence data were obtained from GenBank, EMBL, and SWISSPROTdatabases. Genes were designated as appeared in the references.

TABLE 19 Strains analyzed in Example 43. 16S rDNA sequence accessionTaxon Strain* Strain† number Cedecea davisae ATCC 33431^(|) Cedecealapagei ATCC 33432^(|) Cedecea neteri ATCC 33855^(|) Citrobacteramalonaticus ATCC 25405^(|) CDC 9020-77^(|) AF025370 Citrobacter braakiiATCC 43162 CDC 080-58^(|) AF025368 Citrobacter farmeri ATCC 51112^(|)CDC 2991-81^(|) AF025371 Citrobacter freundii ATCC 8090^(|) DSM30039^(|) AJ233408 Citrobacter koseri ATCC 27156^(|) Citrobactersedlakii ATCC 51115^(|) CDC 4696-86^(|) AF025364 Citrobacter werkmaniiATCC 51114^(|) CDC 0876-58^(|) AF025373 Citrobacter youngae ATCC29935^(|) Edwardsiella hoshinae ATCC 33379^(|) Edwardsiella tarda ATCC15947^(|) CDC 4411-68 AF015259 Enterobacter aerogenes ATCC 13048^(|) JCM1235^(|) AB004750 Enterobacter agglomerans ATCC 27989 Enterobacteramnigenus ATCC 33072^(|) JCM 1237^(|) AB004749 Enterobacter asburiaeATCC 35953^(|) JCM 6051^(|) AB004744 Enterobacter cancerogenus ATCC35317^(|) Enterobacter cloacae ATCC 13047^(|) Enterobacter gergoviaeATCC 33028^(|) JCM 1234^(|) AB004748 Enterobacter hormaechei ATCC49162^(|) Enterobacter sakazakii ATCC 29544^(|) JCM 1233^(|) AB004746Escherichia coli ATCC 11775^(|) ATCC 11775^(|) X80725 Escherichia coliATCC 25922 ATCC 25922 X80724 Escherichia coli (ETEC) ATCC 35401Escherichia coli (O157:H7) ATCC 43895 ATCC 43895 Z83205 Escherichiafergusonii ATCC 35469^(|) Escherichia hermanii ATCC 33650^(|)Escherichia vulneris ATCC 33821^(|) ATCC 33821^(|) X80734 Ewingellaamericana ATCC 33852^(|) NCPPB 3905 X88848 Hafnia alvei ATCC 13337^(|)ATCC 13337^(|) M59155 Klebsiella ornithinolytica ATCC 31898 CIP 103.364U78182 Klebsiella oxytoca ATCC 33496 ATCC 13182^(|) U78183 Klebsiellaplanticola ATCC 33531^(|) JCM 7251^(|) AB004755 Klebsiella pneumoniaesubsp. pneumoniae ATCC 13883^(|) DSM 30104^(|) AJ233420 subsp. ozaenaeATCC 11296^(|) ATCC 11296^(|) Y17654 subsp. rhinoscleromatis ATCC13884^(|) Kluyvera ascorbata ATCC 33433^(|) ATCC 14236 Y07650 Kluyveracryocrescens ATCC 33435^(|) Kluyvera georgiana ATCC 51603^(|) Leclerciaadecarboxylata ATCC 23216^(|) Leminorella grimontii ATCC 33999^(|) DSM5078^(|) AJ233421 Moellerella wisconsensis ATCC 35017^(|) Morganellamorganii ATCC 25830^(|) Pantoea agglomerans ATCC 27155^(|) DSM 3493^(|)AJ233423 Pantoea dispersa ATCC 14589^(|) Plesiomonas shigelloïdes ATCC14029^(|) Pragia fontium ATCC 49100^(|) DSM 5563^(|) AJ233424 Proteusmirabilis ATCC 25933 Proteus penneri ATCC 33519^(|) Proteus vulgarisATCC 13315^(|) DSM 30118^(|) AJ233425 Providencia alcalifaciens ATCC9886^(|) Providencia rettgeri ATCC 9250 Providencia rustigianii ATCC33673^(|) Providencia stuartii ATCC 33672 Rahnella aquatilis ATCC33071^(|) DSM 4594^(|) AJ233426 Salmonella choleraesuis subsp. arizonaeATCC 13314^(|) subsp. choleraesuis serotype Choleraesuis ATCC 7001serotype Enteritidis‡ ATCC 13076^(|) SE22 SE22 serotype Gallinarum ATCC9184 serotype Heidelberg ATCC 8326 serotype Paratyphi A ATCC 9150serotype Paratyphi B ATCC 8759 serotype Typhi‡ ATCC 10749 St111 U88545serotype Typhimurium‡ ATCC 14028 serotype Virchow ATCC 51955 subsp.diarizonae ATCC 43973^(|) subsp. houtenae ATCC 43974^(|) subsp. indicaATCC 43976^(|) subsp. salamae ATCC 43972^(|) Serratia fonticola DSM4576^(|) DSM 4576^(|) AJ233429 Serratia grimesii ATCC 14460^(|) DSM30063^(|) AJ233430 Serratia liquefaciens ATCC 27592^(|) Serratiamarcescens ATCC 13880^(|) DSM 30121^(|) AJ233431 Serratia odorifera ATCC33077^(|) DSM 4582^(|) AJ233432 Serratia plymuthica DSM 4540^(|) DSM4540^(|) AJ233433 Serratia rubidaea DSM 4480^(|) DSM 4480^(|) AJ233436Shigella boydii ATCC 9207 ATCC 9207 X96965 Shigella dysenteriae ATCC11835 ATCC 13313^(|) X96966 ATCC 25931 X96964 Shigella flexneri ATCC12022 ATCC 12022 X96963 Shigella sonnei ATCC 29930^(|) Tatumella ptyseosATCC 33301^(|) DSM 5000^(|) AJ233437 Trabulsiella guamensis ATCC49490^(|) Yersinia enterocolitica ATCC 9610^(|) ATCC 9610^(|) M59292Yersinia frederiksenii ATCC 33641^(|) Yersinia intermedia ATCC 29909^(|)Yersinia pestis RRB KIMD27 ATCC 19428^(T) X75274 Yersiniapseudotuberculosis ATCC 29833^(|) Yersinia rohdei ATCC 43380^(|)ER-2935^(|) X75276 Shewanella putrefaciens ATCC 8071^(|) Vibrio choleraeATCC 25870 ATCC 14035^(|) X74695 T Type strain *Strains used in thisstudy for sequencing of partial tuf and atpD genes. SEQ ID NOs. for tufand atpD sequences corresponding to the above reference strains aregiven in table 7. †Strains used in other studies for sequencing of 16SrDNA gene. When both strain numbers are on the same row, both strainsare considered to be the same although strain numbers may be different.‡Phylogenetic serotypes considered species by the Bacteriological Code(1990 Revision).

TABLE 20 PCR primer pairs used in this study Primer Nucleotide AmpliconSEQ ID NO. Sequence positions* length (bp) Tuf 6645′-AAYATGATIACIGGIGCIGCICARATGGA- 271-299 884 3′ 6975′-CCIACIGTICKICCRCCYTCRCG-3′ 1132-1156 atpD 5685′-RTIATIGGIGCIGTIRTIGAYGT-3′ 25-47 884 5675′-TCRTCIGCIGGIACRTAIAYIGCYTG-3′ 883-908 7005′-TIRTIGAYGTCGARTTCCCTCARG-3′ 38-61 871 5675′-TCRTCIGCIGGIACRTAIAYIGCYTG-3′ 883-908 *The nucleotide positions givenare for E. coli tuf and atpD sequences (GenBank accession no. AE000410and V00267, respectively). Numbering starts from the first base of theinitiation codon.

TABLE 21Selection of M. catarrhalis-specific primer pairs from SEQ ID NO: 2863¹(466 pb DNA fragment) other than those previously tested². MoraxellaMoraxella cata- cata- Amplicon rrhalis rrhalis Moraxella size ATCC ATCCnonlique- Primer Sequence (bp) 43628 53879 faciens SEQ ID SEQ IDCGCTGACGGCTTGTTTGTACCA 118 +³ + − NO: 118 NO: 2673 from U.S. Pat. No.6,001,564 SEQ ID SEQ ID TGTTTTGAGCTTTTTATTTTTTG NO: 119 NO: 2674 Afrom U.S. Pat. No. 6,001,564 VBmcat1 SEQ ID TGCTTAAGATTCACTCTGCCATT 93 + + − NO: 2675 TT VBmcat2 SEQ ID TAAGTCGCTGACGGCTTGTTT NO: 2676VBmcat3 SEQ ID CCTGCACCACAAGTCATCAT 140 + + − NO: 2677 VBmcat4 SEQ IDAATTCACCAACAATGTCAAAGC NO: 2678 VBmcat5 SEQ ID AATGATAACCAGTCAAGCAAGC219 + + − NO: 2679 VBmcat6 SEQ ID GGTGCATGGTGATTTGTAAAA NO: 2680 VBmcat7SEQ ID GTGTGCGTTCACTTTTACAAAT 160 + + − NO: 2681 VBmcat8 SEQ IDGGTGTTAAGCTGATGATGAGAG NO: 2682 VBmcat9 SEQ ID TGACCATGCACACCCTTATT167 + + − NO: 2683 VBmcat10 SEQ ID TCATTGGGATGAAAGTATCGTT NO: 2684Amplicon size Moraxella Moraxella Moraxella Primer Sequence (bp)lacunata osloensis atlantae SEQ ID SEQ ID CGCTGACGGCTTGTTTGTACCA 118 − −− NO: 118 NO: 2673 from U.S. Pat. No. 6,001,564 SEQ ID SEQ IDTGTTTTGAGCTTTTTATTTTTTG NO: 119 NO: 2674 A from U.S. Pat. No. 6,001,564VBmcat1 SEQ ID TGCTTAAGATTCACTCTGCCATT  93 − − − NO: 2675 TT VBmcat2SEQ ID TAAGTCGCTGACGGCTTGTTT NO: 2676 VBmcat3 SEQ IDCCTGCACCACAAGTCATCAT 140 − − − NO: 2677 VBmcat4 SEQ IDAATTCACCAACAATGTCAAAGC NO: 2678 VBmcat5 SEQ ID AATGATAACCAGTCAAGCAAGC219 − − − NO: 2679 VBmcat6 SEQ ID GGTGCATGGTGATTTGTAAAA NO: 2680 VBmcat7SEQ ID GTGTGCGTTCACTTTTACAAAT 160 − − − NO: 2681 VBmcat8 SEQ IDGGTGTTAAGCTGATGATGAGAG NO: 2682 VBmcat9 SEQ ID TGACCATGCACACCCTTATT 167− − − NO: 2683 VBmcat10 SEQ ID TCATTGGGATGAAAGTATCGTT NO: 2684 AmpliconMoraxella size phenyl- Kingella Kingella Primer Sequence (bp) pyruvicaindologenes kingea SEQ ID SEQ ID CGCTGACGGCTTGTTTGTACCA 118 − − −NO: 118 NO: 2673 from U.S. Pat. No. 6,001,564 SEQ ID SEQ IDTGTTTTGAGCTTTTTATTTTTTG NO: 119 NO: 2674 A from U.S. Pat. No. 6,001,564VBmcat1 SEQ ID TGCTTAAGATTCACTCTGCCATT  93 − − − NO: 2675 TT VBmcat2SEQ ID TAAGTCGCTGACGGCTTGTTT NO: 2676 VBmcat3 SEQ IDCCTGCACCACAAGTCATCAT 140 − − − NO: 2677 VBmcat4 SEQ IDAATTCACCAACAATGTCAAAGC NO: 2678 VBmcat5 SEQ ID AATGATAACCAGTCAAGCAAGC219 − − − NO: 2679 VBmcat6 SEQ ID GGTGCATGGTGATTTGTAAAA NO: 2680 VBmcat7SEQ ID GTGTGCGTTCACTTTTACAAAT 160 − − − NO: 2681 VBmcat8 SEQ IDGGTGTTAAGCTGATGATGAGAG NO: 2682 VBmcat9 SEQ ID TGACCATGCACACCCTTATT 167− − − NO: 2683 VBmcat10 SEQ ID TCATTGGGATGAAAGTATCGTT NO: 2684 AmpliconNeisseria size menin- Neisseria Escherichia Primer Sequence (bp) gitidisgonorrhoeae coli SEQ ID SEQ ID CGCTGACGGCTTGTTTGTACCA 118 − − − NO: 118NO: 2673 from U.S. Pat. No. 6,001,564 SEQ ID SEQ IDTGTTTTGAGCTTTTTATTTTTTG NO: 119 NO: 2674 A from U.S. Pat. No. 6,001,564VBmcat1 SEQ ID TGCTTAAGATTCACTCTGCCATT  93 − − − NO: 2675 TT VBmcat2SEQ ID TAAGTCGCTGACGGCTTGTTT NO: 2676 VBmcat3 SEQ IDCCTGCACCACAAGTCATCAT 140 − − − NO: 2677 VBmcat4 SEQ IDAATTCACCAACAATGTCAAAGC NO: 2678 VBmcat5 SEQ ID AATGATAACCAGTCAAGCAAGC219 − − − NO: 2679 VBmcat6 SEQ ID GGTGCATGGTGATTTGTAAAA NO: 2680 VBmcat7SEQ ID GTGTGCGTTCACTTTTACAAAT 160 − − − NO: 2681 VBmcat8 SEQ IDGGTGTTAAGCTGATGATGAGAG NO: 2682 VBmcat9 SEQ ID TGACCATGCACACCCTTATT 167− − − NO: 2683 VBmcat10 SEQ ID TCATTGGGATGAAAGTATCGTT NO: 2684 Ampliconsize Staphylococcus Primer Sequence (bp) aureus SEQ ID SEQ IDCGCTGACGGCTTGTTTGTACCA 118 − NO: 118 NO: 2673 from U.S. Pat. No.6,001,564 SEQ ID SEQ ID TGTTTTGAGCTTTTTATTTTTTG NO: 119 NO: 2674 Afrom U.S. Pat. No. 6,001,564 VBmcat1 SEQ ID TGCTTAAGATTCACTCTGCCATT  93− NO: 2675 TT VBmcat2 SEQ ID TAAGTCGCTGACGGCTTGTTT NO: 2676 VBmcat3SEQ ID CCTGCACCACAAGTCATCAT 140 − NO: 2677 VBmcat4 SEQ IDAATTCACCAACAATGTCAAAGC NO: 2678 VBmcat5 SEQ ID AATGATAACCAGTCAAGCAAGC219 − NO: 2679 VBmcat6 SEQ ID GGTGCATGGTGATTTGTAAAA NO: 2680 VBmcat7SEQ ID GTGTGCGTTCACTTTTACAAAT 160 − NO: 2681 VBmcat8 SEQ IDGGTGTTAAGCTGATGATGAGAG NO: 2682 VBmcat9 SEQ ID TGACCATGCACACCCTTATT 167− NO: 2683 VBmcat10 SEQ ID TCATTGGGATGAAAGTATCGTT NO: 2684 ¹Previouslydisclosed in U.S. Pat. No. 6,001,564 as SEQ ID NO. 29. ²All PCR assayswere performed with 1 ng of purified genomic DNA by using an annealingtemperature of 55° C. and 30 cycles of amplification. The genomic DNAfrom various bacterial species above was always isolated from referencestrains obtained from ATCC. ³All positive results showed a strongamplification signal with genomic DNA from the target speciesM.catarrhalis.

TABLE 22 Selection of S. epidermidis-specific primer pairs fromSEQ ID NO: 2864¹ (705 pb DNA fragment) other than those previously tested.Staphylo- Staphylo- Amplicon coccus coccus Staphylo- Sequence (all 25size epidermidis, epidermidis, coccus Primer nucleotides) (bp)ATCC 14990 ATCC 12228 capitis SEQ ID SEQ ID ATCAAAAAGTTGGCGAACCTTTTCA125 +³ + − NO: 145 NO: 2685 from U.S. Pat. No. 6,001,564 SEQ ID  SEQ IDCAAAAGAGCGTGGAGAAAAGTATCA NO: 146 NO: 2686 from U.S. Pat. No. 6,001,564VBsep3 SEQ ID CATAGTCTGATTGCTCAAAGTCTTG 208 + + − NO: 2687 VBsep4 SEQ IDGCGAATAGTGAACTACATTCTGTTG + + − NO: 2688 VBsep5 SEQ IDCACGCTCTTTTGCAATTTCCATTGA 208 + + + NO: 2689 VBsep6 SEQ IDGAAGCAAATATTCAAAATGCACCAG + + + NO: 2690 VBsep7 SEQ IDAAAGTCTTTTGCTTCTTCAGATTCA 177 + + NT NO: 2691 VBsep8 SEQ IDGTGTTCACAGGTATGGATGCTCTTA + + NT NO: 2692 + + NT VBsep9 SEQ IDGAGCATCCATACCTGTGAACACAGA 153 + + − NO: 2693 VBsep10 SEQ IDTTTTCCAATTACAAGAGACATCAGT + + NT NO: 2694 + + NT VBsep11 SEQ IDTTTGAATTCGCATGTACTTTGTTTG 135 + + − NO: 2695 VBsep12 SEQ IDCCCCGGGTTCGAAATCGATAAAAAG NO: 2696 Amplicon Staphylo- Staphylo-Staphylo- Sequence (all 25 size coccus coccus coccus Primer nucleotides)(bp) cohnii aureus auricularis SEQ ID SEQ ID ATCAAAAAGTTGGCGAACCTTTTCA125 − − − NO: 145 NO: 2685 from U.S. Pat. No. 6,001,564 SEQ ID SEQ IDCAAAAGAGCGTGGAGAAAAGTATCA NO: 146 NO: 2686 from U.S. Pat. No. 6,001,564VBsep3 SEQ ID CATAGTCTGATTGCTCAAAGTCTTG 208 − − − NO: 2687 VBsep4 SEQ IDGCGAATAGTGAACTACATTCTGTTG − − − NO: 2688 VBsep5 SEQ IDCACGCTCTTTTGCAATTTCCATTGA 208 + + − NO: 2689 VBsep6 SEQ IDGAAGCAAATATTCAAAATGCACCAG + + − NO: 2690 VBsep7 SEQ IDAAAGTCTTTTGCTTCTTCAGATTCA 177 − − − NO: 2691 VBsep8 SEQ IDGTGTTCACAGGTATGGATGCTCTTA NT − NT NO: 2692 NT − NT VBsep9 SEQ IDGAGCATCCATACCTGTGAACACAGA 153 − − − NO: 2693 VBsep10 SEQ IDTTTTCCAATTACAAGAGACATCAGT NT − NT NO: 2694 NT − NT VBsep11 SEQ IDTTTGAATTCGCATGTACTTTGTTTG 135 − − − NO: 2695 VBsep12 SEQ IDCCCCGGGTTCGAAATCGATAAAAAG NO: 2696 Amplicon Staphylo- Sequence (all 25size Staphylo- coccus Staphylo- Primer nucleotides) (bp) coccus hominiscoccus SEQ ID SEQ ID ATCAAAAAGTTGGCGAACCTTTTCA 125 − − − NO: 145NO: 2685 from U.S. Pat. No. 6,001,564 SEQ ID SEQ IDCAAAAGAGCGTGGAGAAAAGTATCA NO: 146 NO: 2686 from U.S. Pat. No. 6,001,564VBsep3 SEQ ID CATAGTCTGATTGCTCAAAGTCTTG 208 + − − NO: 2687 VBsep4 SEQ IDGCGAATAGTGAACTACATTCTGTTG − − − NO: 2688 VBsep5 SEQ IDCACGCTCTTTTGCAATTTCCATTGA 208 + + − NO: 2689 VBsep6 SEQ IDGAAGCAAATATTCAAAATGCACCAG + + − NO: 2690 VBsep7 SEQ IDAAAGTCTTTTGCTTCTTCAGATTCA 177 + − − NO: 2691 VBsep8 SEQ IDGTGTTCACAGGTATGGATGCTCTTA − NT − NO: 2692 − NT − VBsep9 SEQ IDGAGCATCCATACCTGTGAACACAGA 153 + − + NO: 2693 VBsep10 SEQ IDTTTTCCAATTACAAGAGACATCAGT + NT + NO: 2694 − NT − VBsep11 SEQ IDTTTGAATTCGCATGTACTTTGTTTG 135 − − − NO: 2695 VBsep12 SEQ IDCCCCGGGTTCGAAATCGATAAAAAG NO: 2696 Amplicon Staphylo- Staphylo-Sequence (all 25 size Staphylo- coccus coccus Primer nucleotides) (bp)coccus simulans warneri SEQ ID SEQ ID ATCAAAAAGTTGGCGAACCTTTTCA 125 − −− NO: 145 NO: 2685 from U.S. Pat. No. 6,001,564 SEQ ID SEQ IDCAAAAGAGCGTGGAGAAAAGTATCA NO: 146 NO: 2686 from U.S. Pat. No. 6,001,564VBsep3 SEQ ID CATAGTCTGATTGCTCAAAGTCTTG 208 − − − NO: 2687 VBsep4 SEQ IDGCGAATAGTGAACTACATTCTGTTG − − − NO: 2688 VBsep5 SEQ IDCACGCTCTTTTGCAATTTCCATTGA 208 − − − NO: 2689 VBsep6 SEQ IDGAAGCAAATATTCAAAATGCACCAG − − NT NO: 2690 VBsep7 SEQ IDAAAGTCTTTTGCTTCTTCAGATTCA 177 − + − NO: 2691 VBsep8 SEQ IDGTGTTCACAGGTATGGATGCTCTTA − + NT NO: 2692 − − NT VBsep9 SEQ IDGAGCATCCATACCTGTGAACACAGA 153 + − − NO: 2693 VBsep10 SEQ IDTTTTCCAATTACAAGAGACATCAGT − − NT NO: 2694 − − NT VBsep11 SEQ IDTTTGAATTCGCATGTACTTTGTTTG 135 − − − NO: 2695 VBsep12 SEQ IDCCCCGGGTTCGAAATCGATAAAAAG NO: 2696 Amplicon Entero- Entero-Sequence (all 25 size Bacillus coccus coccus Primer nucleotides) (bp)subtilis faecalis faecium SEQ ID SEQ ID ATCAAAAAGTTGGCGAACCTTTTCA 125 −− − NO: 145 NO: 2685 from U.S. Pat. No. 6,001,564 SEQ ID SEQ IDCAAAAGAGCGTGGAGAAAAGTATCA NO: 146 NO: 2686 from U.S. Pat. No. 6,001,564VBsep3 SEQ ID CATAGTCTGATTGCTCAAAGTCTTG 208 − − − NO: 2687 VBsep4 SEQ IDGCGAATAGTGAACTACATTCTGTTG − − − NO: 2688 VBsep5 SEQ IDCACGCTCTTTTGCAATTTCCATTGA 208 − − − NO: 2689 VBsep6 SEQ IDGAAGCAAATATTCAAAATGCACCAG NT NT NT NO: 2690 VBsep7 SEQ IDAAAGTCTTTTGCTTCTTCAGATTCA 177 − − − NO: 2691 VBsep8 SEQ IDGTGTTCACAGGTATGGATGCTCTTA NT NT NT NO: 2692 NT NT NT VBsep9 SEQ IDGAGCATCCATACCTGTGAACACAGA 153 − − − NO: 2693 VBsep10 SEQ IDTTTTCCAATTACAAGAGACATCAGT NT NT NT NO: 2694 NT NT NT VBsep11 SEQ IDTTTGAATTCGCATGTACTTTGTTTG 135 − − − NO: 2695 VBsep12 SEQ IDCCCCGGGTTCGAAATCGATAAAAAG NO: 2696 Amplicon Entero- Listeria Strepto-Sequence (all 25 size coccus monocyto- coccus Primer nucleotides) (bp)gallinarum genes agalactiae SEQ ID SEQ ID ATCAAAAAGTTGGCGAACCTTTTCA 125− − − NO: 145 NO: 2685 from U.S. Pat. No. 6,001,564 SEQ ID SEQ IDCAAAAGAGCGTGGAGAAAAGTATCA NO: 146 NO: 2686 from U.S. Pat. No. 6,001,564VBsep3 SEQ ID CATAGTCTGATTGCTCAAAGTCTTG 208 − − − NO: 2687 VBsep4 SEQ IDGCGAATAGTGAACTACATTCTGTTG − − − NO: 2688 VBsep5 SEQ IDCACGCTCTTTTGCAATTTCCATTGA 208 − − − NO: 2689 VBsep6 SEQ IDGAAGCAAATATTCAAAATGCACCAG NT NT NT NO: 2690 VBsep7 SEQ IDAAAGTCTTTTGCTTCTTCAGATTCA 177 − − − NO: 2691 VBsep8 SEQ IDGTGTTCACAGGTATGGATGCTCTTA NT NT NT NO: 2692 NT NT NT VBsep9 SEQ IDGAGCATCCATACCTGTGAACACAGA 153 − − − NO: 2693 VBsep10 SEQ IDTTTTCCAATTACAAGAGACATCAGT NT NT NT NO: 2694 NT NT NT VBsep11 SEQ IDTTTGAATTCGCATGTACTTTGTTTG 135 − − − NO: 2695 VBsep12 SEQ IDCCCCGGGTTCGAAATCGATAAAAAG NO: 2696 Amplicon Strepto- Strepto- AnnealingSequence (all 25 size coccus coccus temperature² Primer nucleotides)(bp) pneumoniae pyogenes (° C.) SEQ ID  SEQ ID ATCAAAAAGTTGGCGAACCTTTTCA125 − − 55 NO: 145 NO: 2685 from U.S. Pat. No. 6,001,564 SEQ ID  SEQ IDCAAAAGAGCGTGGAGAAAAGTATCA NO: 146 NO: 2686 from U.S. Pat. No. 6,001,564VBsep3 SEQ ID CATAGTCTGATTGCTCAAAGTCTTG 208 − − 55 NO: 2687 VBsep4SEQ ID GCGAATAGTGAACTACATTCTGTTG − − 60 NO: 2688 VBsep5 SEQ IDCACGCTCTTTTGCAATTTCCATTGA 208 − − 55 NO: 2689 VBsep6 SEQ IDGAAGCAAATATTCAAAATGCACCAG NT NT 65 NO: 2690 VBsep7 SEQ IDAAAGTCTTTTGCTTCTTCAGATTCA 177 − − 55 NO: 2691 VBsep8 SEQ IDGTGTTCACAGGTATGGATGCTCTTA NT NT 60 NO: 2692 NT NT 65 VBsep9 SEQ IDGAGCATCCATACCTGTGAACACAGA 153 − − 55 NO: 2693 VBsep10 SEQ IDTTTTCCAATTACAAGAGACATCAGT NT NT 60 NO: 2694 NT NT 65 VBsep11 SEQ IDTTTGAATTCGCATGTACTTTGTTTG 135 − − 55 NO: 2695 VBsep12 SEQ IDCCCCGGGTTCGAAATCGATAAAAAG NO: 2696 ¹Previously disclosed in U.S. Pat.No. 6,001,564 as SEQ ID NO.36. ²All PCR assays were performed with 1 ngof purified genomic DNA by using an annealing temperature of 55 to 65°C. and 30 cycles of amplification. The genomic DNA from the variousbacterial species above was always isolated from reference strainsobtained from ATCC. ³All positive results showed a strong amplificationsignal with genomic DNA from the target species S. epidermidis. Theinstensity of the positive amplification signal with species other thanS. epidermidis was variable. NT = NOT TESTED.

TABLE 23Influence of nucleotide variation(s) on the efficiency of the PCR amplification: Example with SEQ ID NO: 146 from S. epidermidis. Staphyloccusepidermidis ² Staphylococcus Number ATCC 14990 aureus ³ Sequence (all 25of 50° C. 55° C. 50° C. Primer¹ nucleotides) mutation 1 1 0.1 0.01 1SEQ ID NO:  SEQ ATCAAAAAGTTGGCGAACCTTTTCA 0 145 from ID U.S. Pat. NO:No. 2697 6,001,564 SEQ ID NO:  SEQ CAAAAGAGCGTGGAGAAAAGTATCA 0  3+⁴ 3+2+ + − 146 from ID U.S. Pat. NO: No. 2698 6,001,564 VBmut1 SEQ ID NO:2699

1 3+ 3+ 2+ + − VBmut2 SEQ ID NO: 2700

1 3+ 3+ 2+ + − VBmut3 SEQ ID NO: 2701

1 3+ 3+ 2+ + − VBmut4 SEQ ID NO: 2702

1 3+ 3+ 2+ + − VBmut5 SEQ ID NO: 2703

1 3+ 3+ 2+ + − VBmut6 SEQ ID NO: 2704

1 3+ 3+ 2+ + − VBmut7 SEQ ID NO: 2705

1 3+ 3+ 2+ + − VBmut8 SEQ ID NO: 2706

1 3+ 3+ 2+ + − VBmut9 SEQ ID NO: 2707

2 3+ 3+ 2+ + − VBmut10 SEQ ID NO: 2708

2 3+ 3+ 2+ + − VBmut11 SEQ ID NO: 2709

2 3+ 3+ 2+ + − VBmut12 SEQ ID NO: 2710

3 3+ 3+ 2+ + − VBmut13 SEQ ID NO: 2711

4 3+ 2+ + − − ¹All PCR tests were performed with SEQ ID NO: 2697 withoutmodification combined with SEQ ID NO: 2698 or 13 modified versions ofSEQ ID NO: 2698. Boxed nucleotides indicate changes in SEQ ID NO: 2698.SEQ ID NOs. 2697 and 2698 were previously disclosed in U.S. Pat. No.6,001,564. ²The tests with S. epidermidis were performed by using anannealing temperature of 55° C. with 1, 0.1 and 0.01 ng of purifiedgenomic DNA or at 50° C. with 1 ng of purified genomic DNA. ³The testswith S. aureus were performed only at 50° C. with 1 ng of genomic DNA.⁴The intensity of the positive amplification signal was quantified asfollows: 3+ = strong signal, 2+ = intermediate signal and + = weaksignal.

TABLE 24Effect of the primer length on the efficiency of the PCR amplification¹:Example with the AT-rich SEQ ID NO: 2714² and SEQ ID NO:2715² from S. epidermidis. Staphylococcus epidermidis ³ ATCC 14990Length 45° C. 55° C. Primer Sequence (nt) 1 0.1 0.01 1 0.1 0.01 VBsep301SEQ ID NO: 2712 ATATCATCAAAAAGTTGGCGAACCTTTTCA 30 NT NT NT 4+ 3+ 2+VBsep302 SEQ ID NO: 2713 AATTGCAAAAGAGCGTGGAGAAAAGTATCA 30 SEQ ID SEQ ID NO: 2714      ATCAAAAAGTTGGCGAACCTTTTCA 25 4+⁵ 3+ 2+ 4+ 3+ 2+NO: 145 from U.S.   Pat. No. 6,001,564 SEQ ID SEQ ID NO: 2715     CAAAAGAGCGTGGAGAAAAGTATCA 25 NO: 146 from U.S. Pat. No. 6,001,564VBsep201 SEQ ID NO: 2716           AAAGTTGGCGAACCTTTTCA 20 NT NT NT 4+3+ 2+ VBsep202 SEQ ID NO: 2717           GAGCGTGGAGAAAAGTATCA 20VBsep171 SEQ ID NO: 2718              GTTGGCGAACCTTTTCA 17 4+ 3+ 2+ 3+2+ + VBsep172 SEQ ID NO: 2719              CGTGGAGAAAAGTATCA 17 VBsep151SEQ ID NO: 2720                TGGCGAACCTTTTCA 15 3+ 2+ + − − − VBsep152SEQ ID NO: 2721                TGGAGAAAAGTATCA 15 StaphylococcusStaphylococcus Staphylococcus Staphylococcus aureus ⁴ haemolyticuscapitis warneri Primer 45 55 45 55 45 55 45 55 VBsep301 SEQ ID NO: 2712NT − NT − NT − NT − VBsep302 SEQ ID NO: 2713 SEQ ID  SEQ ID NO: 2714 − −− − + − − − NO: 145 from U.S. Pat. No. 6,001,564 SEQ ID SEQ ID NO: 2715NO: 146 from U.S. Pat. No. 6,001,564 VBsep201 SEQ ID NO: 2716 NT − NT −NT − NT − VBsep202 SEQ ID NO: 2717 VBsep171 SEQ ID NO: 2718 − − − − − −− − VBsep172 SEQ ID NO: 2719 VBsep151 SEQ ID NO: 2720 − − − − − − − −VBsep152 SEQ ID NO: 2721 ¹All PCR tests were performed using anannealing temperature of 45 or 55° C. and 30 cycles of amplification.²All SEQ ID NOs. in this Table are from U.S. Pat. No. 6,001,546. ³Thetests with S. epidermidis were made with 1, 0.1 and 0.01 ng of purifiedgenomic DNA. ⁴The tests with all other bacterial species were made onlywith 1 ng of purified genomic DNA. ⁵The intensity of the positiveamplification signal was quantified as follows: 4+ = very strong signal,3+ = strong signal, 2+ = intermediate signal and + = weak signal. NT =not tested.

TABLE 25Effect of the primer length on the efficiency of the PCR amplification¹:Example with the GC-rich SEQ ID NO: 2722² and SEQ ID NO:2723² from P. aeruginosa. Pseudomonas aeruginosa ³ Length ATCC 35554Pseudomonas Burkholderia Primer Sequence (nt) 1 0.1 0.01 fluorescens ⁴cepacia SEQ ID NO 83 SEQ ID NO: 2722 CGAGCGGGTGGTGTTCATC 19 2+⁵ + − − −from U.S. Pat. No. 6,001,564 SEQ ID NO 84 SEQ ID NO: 2723CAAGTCGTCGTCGGAGGGA 19 from U.S. Pat. No. 6,001,564 Pse554-16aSEQ ID NO: 2724 CGAGCGGGTGGTGTTC 16 2+ + − − − Pse674-16aSEQ ID NO: 2725 GTCGTCGTCGGAGGGA 16 Pse554-13b SEQ ID NO: 2726GCGGGTGGTGTTC 13 2+ + − − − Pse674-13a SEQ ID NO: 2727 GTCGTCGGAGGGA 13Shewanella Stenotrophomonas Neisseria Haemophilus Primer putidamaltophilia meningitidis parahaemolyticus SEQ ID NO 83 SEQ ID NO: 2722 −− − − from U.S. Pat. No. 6,001,564 SEQ ID NO 84 SEQ ID NO: 2723from U.S. Pat. No. 6,001,564 Pse554-16a SEQ ID NO: 2724 − − − −Pse674-16a SEQ ID NO: 2725 Pse554-13b SEQ ID NO: 2726 − − − − Pse674-13aSEQ ID NO: 2727 ¹All PCR tests were performed using an annealingtemperature of 55° C. and 30 cycles of amplification. ²SEQ ID NOs. 2722and 2723 were previously disclosed in U.S. Pat. No. 6,001,546. ³Thetests with P. aeruginosa were made with 1, 0.1 and 0.01 ng of purifiedgenomic DNA. ⁴The tests with all other bacterial species were made onlywith 1 ng of purified genomic DNA. ⁵The intensity of the positiveamplification signal was quantified as follows: 2+ = strong signal and += moderately strong signal.

TABLE 26 Sequences used for the consensus of the vanA gene GenBankOrganism access number Source E. faecium M97297 E. faecium AX111560Sequence 2293 of patent WO0123604 E. faecium AX085668 Sequence 21 ofpatent WO0112803 E. faecium AX085648 Sequence 1 of patent WO0112803 E.faecium AX110408 Sequence 1141 of patent WO0123604 E. faecium AX110406Sequence 1139 of patent WO0123604 E. faecium AX110320 Sequence 1053 ofpatent WO0123604. E. faecium AX110319 Sequence 1052 of patent WO0123604E. faecium AX110318 Sequence 1051 of patent WO0123604 E. faeciumAX110316 Sequence 1049 of patent WO0123604 E. faecalis AX110321 Sequence1054 of patent WO0123604 E. gallinarum AX110317 Sequence 1050 of patentWO0123604 E. gallinarum AX110322 Sequence 1055 of patent WO0123604. E.flavescens AX110324 Sequence 1057 of patent WO0123604. S. aureusAE017171.1 Direct deposit to NCBI (VRSA)

TABLE 27 Sequences used for the consensus of vanB gene GenBank Organismaccess number Source E. faecium AF310954.1 Tn5382 transposon ligaseVanB2 SLH475 (vanB2) gene E. faecium AY145441.1 D-alanine:D-lactateligase (vanB2) UI709 E. faecium AF310953.1 Tn5382 ligase VanB2 (vanB2)gene VRE-1 E. faecium U94526.1 vancomycin resistance protein B (vanB)gene, E. faecium AJ306727.1 plasmid pVREM3123/00 partial vanHB gene andpartial vanB2 gene E. faecium AJ306726.1 plasmid pVREM2497/00 partialvanHB gene and partial vanB2 gene E. faecium Z83305.1 vanB2, vanHB2 andvanXB2 genes E. faecium Z83305.1 vanB2, vanHB2 and vanXB2 genes E.faecium AF310957.1 transposon Tn5382 ligase VanB2 CG4248 (vanB2) gene,E. faecalis L15304.1 vancomycin resistance vanB2 gene E. faecalisU35369.1 vancomycin resistance genes, response regulator (vanRB),protein histidine kinase (vanSB), D,D-carboxypeptidase (vanYB), putativeD-2-hydroxyacid dehydrogenase (vanHB), D-Ala:D-Lac ligase (vanB), andputative D,D- dipeptidase (vanXB) genes E. faecalis U00456.1 vanB geneE. faecalis L06138.1 VANB gene E. faecalis U72704.1 vancomycinresistance protein (vanB) gene E. faecalis AF310955.1 transposon Tn5382ligase VanB2 T4059 (vanB2) gene E. faecalis L15304.1 vancomycinresistance vanB2 gene E. faecalis AF310955.1 transposon Tn5382 ligaseVanB2 T4059 (vanB2) gene E. faecalis AF192329.1 transposon Tn1549,complete sequence S. bovis Z70527.1 partial vanB2 gene biotype II S.lutetiensis AY035703.1 Tn5382-like transposon ligase strain 5-F9 VanB2(vanB2) gene *Deposited directly to NCBI

TABLE 28 Number of positive assay results out of five in VanR Assay. DNAcopies number E. faecium E. faecalis 0.5 2 2 2.5 5 3 5 5 4 10 5 5 20 5 5

TABLE 29 First list of exemplary strains tested with VanR assay. ATCC orNo Strains Ref. number 1 Enterococcus casseliflavus 25788 2 Enterococcuscecorum 43198 3 Enterococcus columbae 51263 4 Enterococcus malodoratus43197 5 Enterococcus mundtii 43186 6 Enterococcus pseudoavium 49372 7Enterococcus raffinosus 49427 8 Enterococcus dispar 51266 9 Enterococcusdurans 19432 10 Enterococcus faecalis 19433 11 Enterococcus faecalis29212 12 Enterococcus faecalis R830 13 Enterococcus faecium 19434 14Enterococcus faecium N97-330 15 Enterococcus flavescens 49996 16Enterococcus gallinarum 49573 17 Enterococcus saccharolyticus 43076 18Enterococcus sulfureus 49903 19 Enterococcus faecium (vanD) CCRI-1488920 Enterococcus faecium (vanD) CCRI-14889 21 Enterococcus faecalis(vanE) CCRI-1908  22 Enterococcus faecalis (vanE) CCRI-1908  23Enterococcus faecalis (vanG) CCRI-12848 24 Enterococcus faecalis (vanG)CCRI-12848

TABLE 30 Second list of exemplary strains tested with VanR assay ATCC orNo Strains Ref. number 1 Acinetobacter lwoffii CDCF 3697 2 Aeromonashydrophila 7966 3 Bacteroides fragilis 25285 4 Bacillus cereus 13472 5Bifidobacterium breve 15700 6 Candida albicans 10231 7 Ciostridiumdifficile 9689 8 Ciostridium perfringens 13124 9 Corynebacterium bovis7715 10 Escherichia coli 25922 11 Escherichia coli 23511 12Fusobacterium nucleatum 10953 13 Gardnerella vaginalis 14019 14Klebsiella oxytoca 33496 15 Klebsiella pneumoniae 13883 16 Listeriamonocytogenes L 374 17 Morganella morganii subsp. morganii 25830 18Peptostreptococcus anaerobius 27337 19 Peptostreptococcusasaccharolyticus LSPQ 2639 20 Porphyromonas asaccharolytica 25260 21Prevotella melaninogenica 25845 22 Propionibacterium acnes 6919 23Staphylococcus epidermidis 14990 24 Stenotrophomonas maltophilia 13637

TABLE 31 Third list of exemplary strains tested with VanR assay. ATCC orNo Strains Ref. number 1 Abiotrophia defectiva 49176 2 Acinetobacterbaumannii 19606 3 Actinomyces pyogenes 19411 4 Citrobacter braakii 431625 Citrobacter koseri 27028 6 Corynebacterium genitalium LSPQ 3583 7Enterobacter cloacae 13047 8 Hafnia alvei 13337 9 Homo sapiens 2.16 10Lactobacillus acidophilus 4356 11 Lactobacillus gasseri 33323 12Mobiluncus curtisii subsp. holmesii 35242 13 Neisseria gonorrhoeae 3520114 Pantoea agglomerans 27155 15 Pseudomonas aeruginosa 35554 16Salmonella enterica subsp. Arizonae 13314 17 Salmonella enterica subsp.Enterica 7001 18 Salmonella typhimurium 14028 19 Shigella flexneri 1202220 Shigella sonnei 29930 21 Staphylococcus aureus 43300 22 Streptococcusanginosus 33397 23 Streptococcus bovis 33317 24 Vibrio cholerae 25870 25Yersinia enterocolitica subs. enterolitica 23715

TABLE 32 Vancomycin resistant strains tested with VanR assay at 10⁵CFU/rx. Resistance ATCC or No Strains gene Ref. number 1-2 Enterococcuscasseliflavus vanC2 25788 (10⁴ CFU/rx) 3-4 Enterococcus casseliflavusvanC2 25788 5-6 Enterococcus faecalis vanG  CCRI-12849 7-8 Enterococcusfaecium vanD R832  9-10 Enterococcus faecium vanD2  CCRI-15140 11-12Enterococcus faecium vanD CCRI-2062 13-14 Enterococcus faecium vanDCCRI-8824 15-16 Enterococcus gallinarum vanC CCRI-9133 17-18Enterococcus gallinarum vanC CCRI-9131 19-20 Enterococcus gallinarumvanA and CCRI-1561 vanC1

TABLE 33 Composition of premix (PM) CONCENTRATION IN FRESHLY PREPAREDMIX (28.8 μL COMPONENT FINAL VOLUME) FastStart Taq DNA Polymerase 0.060U/μL MgCl₂ 3.010 mM Tris 100.333 mM KCl 10.033 mM (NH₄)₂SO₄ 5.017 mMdNTPs 200.667 μM Primer vanA649 (SEQ ID NO: 1090) 0.120 μM PrimervanA754 (SEQ IDNO: 1091) 0.101 μM Primer vanB626 (SEQ ID NO: 2298) 0.702μM Primer vanB774 (SEQ ID NO: 1096) 0.702 μM Probe VanA-B5c-A0 (SEQ IDNO: 2299) 0.120 μM Probe VanB-B50-F0 (SEW ID NO: 2300) 0.351 μM ProbeSign-B4-B0 (SEQ ID NO: 2301) 0.201 μM pERVd Internal control (SEQ ID NO:2302) 3.582 copies/μL (usual) S. epidermidis DNA 895.836 copies/μL BSA0.301 mg/mL Trehalose 4.014% *These components were added followingTable 21

TABLE 34 Composition of final mix (MM) Sign- VanA- VanB- IC B4 B5c B50(SEQ (SEQ ID (SEQ ID (SEQ ID ID NO: n NO: 2301) NO: 2299) NO: 2300)2302) Specimen MM1-a 10 x DNA³ MM1-b 5 x x¹ TE 1X MM2-a 10 X DNA³ MM2-b5 X x¹ TE 1X MM3-a 10 x DNA³ MM3-b 5 X x¹ TE 1X MM4-std 15 x X X x²DNA⁴/ TE 1X ¹At 100 times the usual concentration ²At usualconcentration ³At 25/50 VanA/VanB DNA copies ⁴At 1 ng/rx VanA/VanB DNAcopies

TABLE 35 Positive results obtained in indicated beacon channel Sign-B4VanA-B5 VanB-B50 (SEQ ID NO: (SEQ ID NO: (SEQ IDNO: 2301) TET 2299)c FAM2300) Texas Red Target MM1-a 0/5 — — Efm 0/5 — — Efs MM1-b 5/5 — — ICMM2-a — 5/5 — Efm — 0/5 — Efs MM2-b — 0/5 — IC MM3-a — — 0/5 Efm — — 5/5Efs MM3-b — — 0/5 IC MM4-std 5/5 5/5 0/5 Efm 5/5 0/5 5/5 Efs 5/5 0/5 0/5IC

TABLE 36 List of enterococcal strains from different locations aroundthe world tested with VanR assay (dilutions 10⁻³). Reference vanA vanBNo Strains number Location CT CT 1 Enterococcus faecalis CCRI-1471Texas, USA 29.91 — 2 Enterococcus faecalis CCRI-1528 Quebec, 27.05 — CAN3 Enterococcus faecalis CCRI-9741 USA — 31.05 4 Enterococcus gallinarumCCRI-1568 Quebec, 28.60 — CAN 5 Enterococcus faecium CCRI-9911 Taiwan, —31.96 China 6 Enterococcus faecalis  725 Israel — 31.55 7 Enterococcusfaecalis 1435 Brazil 28.28 — 8 Enterococcus faecium 6169 Italy 31.63 — 9Enterococcus gallinarum CCRI-9737 Norway — 29.50 10 Enterococcusfaecalis CCRI-9738 Germany — 31.35 11 Enterococcus faecium CCRI-9740Germany — 29.41 12 Enterococcus faecalis 13024  Germany 27.49 — 13Enterococcus faecalis CCRI-9739 Germany — 31.00 14 Enterococcus faeciumCCRI-9733 USA — 31.93 15 Enterococcus faecium 1585 Argentina 29.00 — 16Enterococcus faecium  715 Sweden — 30.87 17 Enterococcus faecalisCCRI-9954 Netherlands — 31.83 18 Enterococcus faecium CCRI-1482 Toronto,29.40 — CAN *All strains gave vanA or vanB positive PCR results.

TABLE 37 List of resistant non enterococcal strains (vanB genotype)tested with VanR assay. Reference Texas Red CT No Strains number(cycles) 1 Clostridium innocuum CCRI-9927 29.6 2 Clostridium sp.IDI-1987 31.9 3 Clostridium sp. CCRI-9929 32.5 4 Clostridium symbosiumCCRI-9928 31.6 5 Eggerthella lenta CCRI-9926 32.4 *All strains gave vanBpositive PCR results.

TABLE 38 VanR Assay Formulation Concentration of Concentration ofcomponents at final volume components at final volume Raw Material (28μL) Lyophilized (28.8 μL) Fresh FastStart Taq DNA Polymerase 0..060U/μL¹ 0.060 U/μL¹ MgCl₂ 3.000 mM² 3.010 mM² Tris 100.000 mM (pH 8.3)²100.333 mM (pH 8.3)² KCl 10.000 mM² 10.033 mM² (NH₄)₂SO₄ 5.000 mM² 5.017mM² dNTP 200.000 μM 200.667 μM Primer VanA 649 (SEQ ID NO: 1090) 0.120μM 0.120 μM Primer Van A 754 (SEQ ID NO: 1091) 0.100 μM 0.101 μM PrimerVan B 626 (SEQ ID NO: 2298) 0.700 μM 0.702 μM Primer van B 774 (SEQ IDNO: 1096) 0.700 μM 0.702 μM Probe VanA-B5c-A0 (SEQ ID NO: 2299) 0.120 μM0.120 μM Probe VanB-B50-F0 (SEQ ID NO: 2300) 0.350 μM 0.351 μM ProbeSign-B4-B0 (SEQ ID NO: 2301) 0.200 μM 0.201 μM Internal control pERVd(SEQ ID NO: 2302) 3.570 copies/μL 3.582 copies/μL S. epidermidis DNA892.857 copies/μL³ 895.836 copies/μL³ BSA 0.300 mg/mL 0.301 mg/mLTrehalose 4.000% 4.014%

TABLE 39 Specific and ubiquitous primers for nucleic acidamplification (tuf sequences). Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionBacterial species: Acinetobacter baumannii 1692 5′-GGT GAG AAC TGT GGT ATC TTA CTT   1 478-501  1693^(a)5′-CAT TTC AAC GCC TTC TTT CAA CTG   1 691-714 Bacterial species: Chlamydia pneumoniae 6305′-CGG AGC TAT CCT AGT CGT TTC A  20  2-23  629^(a)5′-AAG TTC CAT CTC AAC AAG GTC AAT A  20 146-170  2085 5′-CAA ACT AAA GAA CAT ATC TTG CTA  20 45-68  2086^(a)5′-ATA TAA TTT GCA TCA CCT TCA AG  20 237-259  2087 5′-TCA GCT CGT GGG ATT AGG AGA G  20 431-452  2088^(a)5′-AGG CTT CAC GCT GTT AGG CTG A  20 584-605 Bacterial species: Chlamydia trachomatis 5545′-GTT CCT TAC ATC GTT GTT TTT CTC  22  82-105  555^(a)5′-TCT CGA ACT TTC TCT ATG TAT GCA  22 249-272 Parasitical species: Cryptosporidium parvum 7985′-TGG TTG TCC CAG CCG ATC GTT T 865 158-179   804^(a)5′-CCT GGG ACG GCC TCT GGC AT 865 664-683  7995′-ACC TGT GAA TAC AAG CAA TCT 865 280-300   805^(a)5′-CTC TTG TCC ATC TTA GCA GT 865 895-914  8005′-GAT GAA ATC TTC AAC GAA GTT GAT 865 307-330   806^(a)5′-AGC ATC ACC AGA CTT GAT AAG 865 946-966  8015′-ACA ACA CCG AGA AGA TCC CA 865 353-372   803^(a)5′-ACT TCA GTG GTA ACA CCA GC 865 616-635  8025′-TTG CCA TTT CTG GTT TCG TT 865 377-396   807^(a)5′-AAA GTG GCT TCA AAG GTT GC 865  981-1000Bacterial species: Enterococcus faecium 1696 5′-ATG TTC CTG TAG TTG CTG GA  64 189-208  1697^(a)5′-TTT CTT CAG CAA TAC CAA CAA C  64 422-443 Bacterial species: Klebsiella pneumoniae 1329 5′-TGT AGA GCG CGG TAT CAT CAA AGT A 103 352-377  1330^(a)5′-AGA TTC GAA CTT GGT GTG CGG G 103 559-571 ^(a)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.Bacterial species: Mycoplasma pneumoniae 2093 5′-TGT TGG CAA TCG AAG ACA CC 2097^(a) 635-654  2094^(b)5′-TTC AAT TTC TTG ACC TAC TTT CAA 2097^(a) 709-732 Bacterial species: Neisseria gonorrhoeae 5515′-GAA GAA AAA ATC TTC GAA CTG GCT A 126 256-280   552^(b)5′-TAC ACG GCC GGT GAC TAC G 126 378-396  2173 5′-AAG AAA AAA TCT TCG AAC TGG CTA 126 257-280  2174^(b)5′-TCT ACA CGG CCG GTG 126 384-398  2175  5′-CCG CCA TAC CCC GTT T 126654-669  2176^(b) 5′-CGG CAT TAC CAT TTC CAC ACC TTT 126 736-759 Bacterial species: Pseudomonas aeruginosa 1694 5′-AAG GCA AGG ATG ACA ACG GC 153 231-250  1695^(b)5′-ACG ATT TCC ACT TCT TCC TGG 153 418-438 Bacterial species: Streptococcus agalactiae 5495′-GAA CGT GAT ACT GAC AAA CCT TTA 207-210^(c)  308-331^(d)  550^(b)5′-GAA GAA GAA CAC CAA CGT TG 207-210^(c)  520-539^(d)Bacterial species: Streptococcus pyogenes 9995′-TTG ACC TTG TTG ATG ACG AAG AG 1002  143-165  1000^(b)5′-TTA GTG TGT GGG TTG ATT GAA CT 1002  622-644  1001 5′-AAG AGT TGC TTG AAT TAG TTG AG 1002  161-183  1000^(b)5′-TTA GTG TGT GGG TTG ATT GAA CT 1002  622-644 Parasitical species: Trypanosoma brucei 8205′-GAA GGA GGT GTC TGC TTA CAC 864 513-533   821^(b)5′-GGC GCA AAC GTC ACC ACA TCA 864 789-809  8205′-GAA GGA GGT GTC TGC TTA CAC 864 513-533   822^(b)5′-CGG CGG ATG TCC TTA ACA GAA 864 909-929  ^(a)Sequence from databases.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(c)These sequences were aligned to derive the corresponding primer.^(d)The nucleotide positions refer to the S. agalactiae tuf sequence fragment(SEQ ID NO. 209). Parasitical species: Trypanosoma cruzi 7945′-GAC GAC AAG TCG GTG AAC TT 840-842^(a)  281-300^(c)  795^(b)5′-ACT TGC ACG CGA TGT GGC AG 840-842^(a)  874-893^(c)Bacterial genus: Clostridium sp. 796 5′-GGT CCA ATG CCW CAA ACW AGA32, 719-724,  32-52^(d) 736^(a)  797^(b)5′-CAT TAA GAA TGG YTT ATC TGT SKC TCT 32, 719-724,  320-346^(d) 736^(a)808 5′-GCI TTA IWR GCA TTA GAA RAY CCA 32, 719-724,  224-247^(d) 736^(a) 809^(b) 5′-TCT TCC TGT WGC AAC TGT TCC TCT 32, 719-724,  337-360^(d)736^(a) 810 5′-AGA GMW ACA GAT AAR SCA TTC TTA 32, 719-724,  320-343^(d)736^(a)  811^(b) 5′-TRA ART AGA ATT GTG GTC TRT ATC C 32, 719-724, 686-710^(d) 736^(a) Bacterial genus: Corynebacterium sp. 5455′-TAC ATC CTB GTY GCI CTI AAC AAG TG 34-44, 662^(a)   89-114^(e) 546^(b) 5′-CCR CGI CCG GTR ATG GTG AAG AT 34-44, 662^(a)  350-372^(e)Bacterial genus: Enterococcus sp. 656 5′-AAT TAA TGG CTG CAG TTG AYG A58-72^(a)  273-294^(f)  657^(b) 5′-TTG TCC ACG TTC GAT RTC TTC A58-72^(a)  556-577^(f) 656 5′-AAT TAA TGG CTG CAG TTG AYG A 58-72^(a) 273-294^(f)  271^(b) 5′-TTG TCC ACG TTG GAT RTC TTC A 58-72^(a) 556-577^(f) 1137  5′-AAT TAA TGG CTG CWG TTG AYG AA 58-72^(a) 273-295^(f) 1136^(b) 5′-ACT TGT CCA CGT TSG ATR TCT 58-72^(a) 559-579^(f)^(a)These sequences were aligned to derive the corresponding primer.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(c)The nucleotide positions refer to the T. cruzi tuf sequence fragment (SEQ IDNO. 842).^(d)The nucleotide positions refer to the C. perfringens tuf sequence fragment(SEQ ID NO. 32).^(e)The nucleotide positions refer to the C. diphtheriae tuf sequence fragment(SEQ ID NO. 662).^(f)The nucleotide positions refer to the E. durans tuf sequence fragment (SEQ IDNO. 61). Bacterial genus: Legionella sp. 2081 5′-GRA TYR TYA AAG TTG GTG AGG AAG 111-112^(a)  411-434^(b) 2082^(c)5′-CMA CTT CAT CYC GCT TCG TAC C 111-112^(a)  548-569^(b)Bacterial genus: Staphylococcus sp. 5535′-GGC CGT GTT GAA CGT GGT CAA ATC A 176-203^(a)  313-337^(d)  575^(c)5′-TIA CCA TTT CAG TAC CTT CTG GTA A 176-203^(a)  653-677^(d) 5535′-GGC CGT GTT GAA CGT GGT CAA ATC A 176-203^(a)  313-337^(d)  707^(c)5′-TWA CCA TTT CAG TAC CTT CTG GTA A 176-203^(a)  653-677^(d)Bacterial genus: Streptococcus sp. 547 5′-GTA CAG TTG CTT CAG GAC GTA TC206-231^(a)  372-394^(e)  548^(c) 5′-ACG TTC GAT TTC ATC ACG TTG206-231^(a)  548-568^(e) Fungal genus: Candida sp. 5765′-AAC TTC RTC AAG AAG GTY GGT TAC AA 407-426,  332-357^(f) 428-432^(a) 632^(c) 5′-CCC TTT GGT GGR TCS TKC TTG GA 407-426,  791-813^(f)428-432^(a) 631 5′-CAG ACC AAC YGA IAA RCC ATT RAG AT 407-426, 523-548^(f) 428-432^(a)  632^(c) 5′-CCC TTT GGT GGR TCS TKC TTG GA407-426,  791-813^(f) 428-432^(a) 6335′-CAG ACC AAC YGA IAA RCC ITT RAG AT 407-426,  523-548^(f) 428-432^(a) 632^(c) 5′-CCC TTT GGT GGR TCS TKC TTG GA 407-426,  791-813^(f)428-432^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the L. pneumophila tuf sequence fragment(SEQ ID NO. 112).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the S. aureus tuf sequence fragment (SEQ IDNO. 179).^(e)The nucleotide positions refer to the S. agalactiae tuf sequence fragment(SEQ ID NO. 209).^(f)The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment(SEQ ID NO. 408). Fungal genus: Cryptococcus sp. 1971 5′-CYG ACT GYG CCA TCC TYA TCA 434, 623, 1281, 150-170^(b)1985, 1986^(a) 1973^(c) 5′-RAC ACC RGI YTT GGW ITC CTT 434, 623, 1281,464-484^(b) 1985, 1986^(a) 1972  5′-MGI CAG CTC ATY ITT GCW KSC434, 623, 1281, 260-280^(b) 1985, 1986^(a) 1973^(c)5′-RAC ACC RGI YTT GGW ITC CTT 434, 623, 1281, 464-484^(b)1985, 1986^(a) Parasitical genus: Entamoeba sp. 7035′-TAT GGA AAT TCG AAA CAT CT 512 38-57   704^(c)5′-AGT GCT CCA ATT AAT GTT GG 512 442-461  7035′-TAT GGA AAT TCG AAA CAT CT 512 38-57   705^(c)5′-GTA CAG TTC CAA TAC CTG AA 512 534-553  7035′-TAT GGA AAT TCG AAA CAT CT 512 38-57   706^(c)5′-TGA AAT CTT CAC ATC CAA CA 512 768-787  7935′-TTA TTG TTG CTG CTG GTA CT 512 149-168   704^(c)5′-AGT GCT CCA ATT AAT GTT GG 512 442-461 Parasitical genus: Giardia sp. 816 5′-GCT ACG ACG AGA TCA AGG GC 513305-324   819^(c) 5′-TCG AGC TTC TGG AGG AAG AG 513 895-914  8175′-TGG AAG AAG GCC GAG GAG TT 513 355-374   818^(c)5′-AGC CGG GCT GGA TCT TCT TC 513 825-844 Parasitical genus: Leishmania sp. 701 5′-GTG TTC ACG ATC ATC GAT GCG514-526^(a)  94-114^(d)  702^(c) 5′-CTC TCG ATA TCC GCG AAG CG514-526^(a) 913-932^(d)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the C. neoformans tuf (EF-1) sequencefragment (SEQ ID NO. 623).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the L. tropica tuf(EF-1) sequence fragment(SEQ ID NO. 526). Parasitical genus: Trypanosoma sp. 8235′-GAG CGG TAT GAY GAG ATT GT 529, 840-842, 493-512^(b) 864^(a)  824^(c)5′-GGC TTC TGC GGC ACC ATG CG 529, 840-842, 1171-1190^(b) 864^(a)Bacterial family: Enterobacteriaceae 9335′-CAT CAT CGT ITT CMT GAA CAA RTG 78, 103, 146, 390-413^(d)168, 238, 698^(a)  934^(c) 5′-TCA CGY TTR RTA CCA CGC AGI AGA78, 103, 146, 831-854^(d) 168, 238, 698^(a)Bacterial family: Mycobacteriaceae 5395′-CCI TAC ATC CTB GTY GCI CTI AAC AAG 122  85-111  540^(c)5′-GGD GCI TCY TCR TCG WAI TCC TG 122 181-203 Bacterial group: Escherichia coli and Shigella 1661 5′-TGG GAA GCG AAA ATC CTG 1668^(e) 283-300  1665^(c)5′-CAG TAC AGG TAG ACT TCT G 1668^(e) 484-502 Bacterial group: Pseudomonads group 5415′-GTK GAA ATG TTC CGC AAG CTG CT 153-155^(a) 476-498^(f)  542^(c)5′-CGG AAR TAG AAC TGS GGA CGG TAG 153-155^(a) 679-702^(f) 5415′-GTK GAA ATG TTC CGC AAG CTG CT 153-155^(a) 476-498^(f)  544^(c)5′-AYG TTG TCG CCM GGC ATT MCC AT 153-155^(a) 749-771^(f)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the T. brucei tuf (EF-1) sequence fragment(SEQ ID NO. 864).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d )The nucleotide positions refer to the E. coli tuf sequence fragment (SEQ IDNO. 698). ^(e)Sequence from databases.^(f)The nucleotide positions refer to the P. aeruginosa tuf sequence fragment(SEQ ID NO. 153). Parasitical group: Trypanosomatidae family 9235′-GAC GCI GCC ATC CTG ATG ATC 511, 514-526, 166-188^(b) 529, 840-842,864^(a)  924^(c) 5′-ACC TCA GTC GTC ACG TTG GCG 511, 514-526,648-668^(b) 529, 840-842, 864^(a) 925 5′-AAG CAG ATG GTT GTG TGC TG511, 514-526, 274-293^(b) 529, 840-842, 864^(a)  926^(c)5′-CAG CTG CTC GTG GTG CAT CTC GAT 511, 514-526, 676-699^(b)529, 840-842, 864^(a) 927 5′-ACG CGG AGA AGG TGC GCT T 511, 514-526,389-407^(b) 529, 840-842, 864^(a)  928^(c)5′-GGT CGT TCT TCG AGT CAC CGC A 511, 514-526, 778-799^(b) 529, 840-842,864^(a) Universal primers (bacteria) 6365′-ACT GGY GTT GAI ATG TTC CGY AA 7, 54, 78, 470-492^(d) 100, 103, 159,209, 224, 227^(b)  637^(c) 5′-ACG TCA GTI GTA CGG AAR TAG AA 7, 54, 78,692-714^(d) 100, 103, 159, 209, 224, 227^(b) 6385′-CCA ATG CCA CAA ACI CGT GAR CAC AT 7, 54, 78, 35-60^(e)100, 103, 159, 209, 224, 227^(b)  639^(c)5′-TTT ACG GAA CAT TTC WAC ACC WGT IAC A 7, 54, 78, 469-496^(e)100, 103, 159, 209, 224, 227^(b)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the L. tropica tuf (EF-1) sequence fragment(SEQ ID NO. 526).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the E. coli tuf sequence fragment (SEQ IDNO. 78).^(e)The nucleotide positions refer to the B. cereus tuf sequence fragment (SEQ IDNO. 7). 643 5′-ACT GGI GTI GAR ATG TTC CGY AA 1, 3, 4, 7, 12,470-492^(b) 13, 16, 49, 54, 72, 78, 85, 88, 91, 94, 98, 103,108, 112, 115, 116, 120, 121, 126, 128, 134, 136, 146, 154,159, 179, 186, 205, 209, 212, 224, 238^(a)  644^(c)5′-ACG TCI GTI GTI CKG AAR TAG AA same as SEQ 692-714^(b) ID NO. 643 6435′-ACT GGI GTI GAR ATG TTC CGY AA 1, 3, 4, 7, 12, 470-492^(b)13, 16, 49, 54, 72, 78, 85, 88, 91, 94, 98, 103, 108, 112, 115,116, 120, 121, 126, 128, 134, 136, 146, 154, 159, 179, 186,205, 209, 212, 224, 238^(a)  645^(c) 5′-ACG TCI GTI GTI CKG AAR TAR AAsame as SEQ 692-714^(b) ID NO. 643 646 5′-ATC GAC AAG CCI TTC YTI ATG SC2, 13, 82 317-339^(d) 122, 145^(a)  647^(c)5′-ACG TCC GTS GTR CGG AAG TAG AAC TG 2, 13, 82 686-711^(d) 122, 145^(a)646 5′-ATC GAC AAG CCI TTC YTI ATG SC 2, 13, 82 317-339^(d) 122, 145^(a) 648^(c) 5′-ACG TCS GTS GTR CGG AAG TAG AAC TG 2, 13, 82 686-711^(d)122, 145^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the E. coli tuf sequence fragment (SEQ IDNO. 78).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the A. meyeri tuf sequence fragment (SEQ IDNO. 2) 649 5′-GTC CTA TGC CTC ARA CWC GIG AGC AC 8, 86, 141, 143^(a)33-58^(b)  650^(c) 5′-TTA CGG AAC ATY TCA ACA CCI GT 8, 86, 141, 143^(a)473-495^(b) 636 5′-ACT GGY GTT GAI ATG TTC CGY AA 8, 86, 141, 143^(a)473-495^(b)  651^(c) 5′-TGA CGA CCA CCI TCY TCY TTY TTC A8, 86, 141, 143^(a) 639-663^(b) Universal primers (fungi) 19745′-ACA AGG GIT GGR MSA AGG AGA C 404, 405, 433, 443-464^(d)445, 898, 1268, 1276, 1986^(a) 1975^(c) 5′-TGR CCR GGG TGG TTR AGG ACG404, 405, 433, 846-866^(d) 445, 898, 1268, 1276, 1986^(a) 1976 5′-GAT GGA YTC YGT YAA ITG GGA 407-412, 286-306^(e) 414-426, 428-431,439, 443, 447, 448, 622, 624, 665, 1685, 1987-1990^(a) 1978^(c)5′-CAT CIT GYA ATG GYA ATC TYA AT same as SEQ 553-575^(e) ID NO. 19761977  5′-GAT GGA YTC YGT YAA RTG GGA same as SEQ 286-306^(e) ID NO. 19761979^(c) 5′-CAT CYT GYA ATG GYA ASC TYA AT same as SEQ 553-575^(e)ID NO. 1976 1981  5′-TGG ACA CCI SCA AGI GGK CYG 401-405, 281-301^(d)433, 435, 436, 438, 444, 445, 449, 453, 455, 457, 779,781-783, 785, 786, 788-790, 897-903, 1267-1272, 1274-1280,1282-1287, 1991-1998^(a) 1980^(c) 5′-TCR ATG GCI TCI AIR AGR GTY Tsame as SEQ 488-509^(d) ID NO. 1981^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the B. distasonis tuf sequence fragment(SEQ ID NO. 8).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the A. fumigatus tuf (EF-1) sequencefragment (SEQ ID NO. 404).^(e)The nucleotide positions refer to the C. albicans tuf (EF-1) sequence fragment(SEQ ID NO. 407). 1982  5′-TGG ACA CYI SCA AGI GGK CYG same as SEQ281-301^(a) ID NO. 1981 1980^(b) 5′-TCR ATG GCI TCI AIR AGR GTY Tsame as SEQ 488-509^(a) ID NO. 1981 1983  5′-CYG AYT GCG CYA TIC TCA TCAsame as SEQ 143-163^(a) ID NO. 1981 1980^(b)5′-TCR ATG GCI TCI AIR AGR GTY T same as SEQ 488-509^(a) ID NO. 19811984  5′-CYG AYT GYG CYA TYC TSA TCA same as SEQ 143-163^(a) ID NO. 19811980^(b) 5′-TCR ATG GCI TCI AIR AGR GTY T same as SEQ 488-509^(a)ID NO. 1981 Sequencing primers 556 5′-CGG CGC NAT CYT SGT TGT TGC668^(c) 306-326   557^(b) 5′-CCM AGG CAT RAC CAT CTC GGT G  668^(c)1047-1068  694 5′-CGG CGC IAT CYT SGT TGT TGC  668^(c) 306-326   557^(b)5′-CCM AGG CAT RAC CAT CTC GGT G  668^(c) 1047-1068  6645′-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA  619^(c) 604-632   652^(b)5′-CCW AYA GTI YKI CCI CCY TCY CTI ATA  619^(c) 1482-1508  6645′-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA  619^(c) 604-632   561^(b)5′-ACI GTI CGG CCR CCC TCA CGG AT  619^(c) 1483-1505  5435′-ATC TTA GTA GTT TCT GCT GCT GA 607  8-30  660^(b)5′-GTA GAA TTG AGG ACG GTA GTT AG 607 678-700  6585′-GAT YTA GTC GAT GAT GAA GAA TT 621 116-138   659^(b)5′-GCT TTT TGI GTT TCW GGT TTR AT 621 443-465  6585′-GAT YTA GTC GAT GAT GAA GAA TT 621 116-138   661^(b)5′-GTA GAA YTG TGG WCG ATA RTT RT 621 678-700  5585′-TCI TTY AAR TAY GCI TGG GT  665^(c) 157-176   559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT  665^(c) 1279-1301  8135′-AAT CYG TYG AAA TGC AYC ACG A  665^(c) 687-708   559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT 665^(c) 1279-1301 ^(a)The nucleotide positions refer to the A. fumigatus tuf (EF-1) sequencefragment (SEQ ID NO. 404).^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(c)Sequences from databases. 558 5′-TCI TTY AAR TAY GCI TGG GT  665^(a)157-176   815^(b) 5′-TGG TGC ATY TCK ACR GAC TT  665^(a) 686-705  5605′-GAY TTC ATY AAR AAY ATG ATY AC  665^(a) 289-311   559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT  665^(a) 1279-1301  6535′-GAY TTC ATI AAR AAY ATG AT  665^(a) 289-308   559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT  665^(a) 1279-1301  5585′-TCI TTY AAR TAY GCI TGG GT  665^(a) 157-176   655^(b)5′-CCR ATA CCI CMR ATY TTG TA  665^(a) 754-773  6545′-TAC AAR ATY KGI GGT ATY GG  665^(a) 754-773   559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT  665^(a) 1279-1301  6965′-ATI GGI CAY RTI GAY CAY GGI AAR AC  698^(a) 52-77   697^(b)5′-CCI ACI GTI CKI CCR CCY TCR CG  698^(a) 1132-1154  9115′-GAC GGM KKC ATG CCG CAR AC 853 22-41   914^(b)5′-GAA RAG CTG CGG RCG RTA GTG 853 700-720  9125′-GAC GGC GKC ATG CCG CAR AC 846 20-39   914^(b)5′-GAA RAG CTG CGG RCG RTA GTG 846 692-712  9135′-GAC GGY SYC ATG CCK CAG AC 843 251-270   915^(b)5′-AAA CGC CTG AGG RCG GTA GTT 843 905-925  9165′-GCC GAG CTG GCC GGC TTC AG 846 422-441   561^(b)5′-ACI GTI CGG CCR CCC TCA CGG AT  619^(a) 1483-1505  6645′-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA  619^(a) 604-632   917^(b)5′-TCG TGC TAC CCG TYG CCG CCA T 846 593-614 ^(a)Sequences from databases.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing. 1221 5′-GAY ACI CCI GGI CAY GTI GAY TT 1230^(a) 292-314  1226^(b)5′-GTI RMR TAI CCR AAC ATY TC 1230^(a) 2014-2033  1222 5′-ATY GAY ACI CCI GGI CAY GTI GAY TT 1230^(a) 289-314  1223^(b)5′-AYI TCI ARR TGI ARY TCR CCC ATI CC 1230^(a) 1408-1433  1224 5′-CCI GYI HTI YTI GAR CCI ATI ATG 1230^(a) 1858-1881  1225^(b)5′-TAI CCR AAC ATY TCI SMI ARI GGI AC 1230^(a) 2002-2027  1227 5′-GTI CCI YTI KCI GAR ATG TTY GGI TA 1230^(a) 2002-2027  1229^(b)5′-TCC ATY TGI GCI GCI CCI GTI ATC AT  698^(a)  4-29 1228 5′-GTI CCI YTI KCI GAR ATG TTY GGI TAY GC 1230^(a) 2002-2030  1229^(b)5′-TCC ATY TGI GCI GCI CCI GTI ATC AT  698^(a)  4-29 1999 5′-CAT GTC AAY ATT GGT ACT ATT GGT CAT GT 498-500,  25-53^(d)502, 505, 506, 508, 619, 2004, 2005^(c) 2000^(b)5′-CCA CCY TCI CTC AMG TTG AAR CGT T same as SEQ 1133-1157^(d)ID NO. 1999 2001  5′-ACY ACI TTR ACI GCY GCY ATY AC same as SEQ 67-89^(d) ID NO. 1999 2003^(b) 5′-CAT YTC RAI RTT GTC ACC TGGsame as SEQ 1072-1092^(d) ID NO. 1999 2002 5′-CCI GAR GAR AGA GCI MGW GGT same as SEQ 151-171^(d) ID NO. 19992003^(b) 5′-CAT YTC RAI RTT GTC ACC TGG same as SEQ 1072-1092^(d)ID NO. 1999 ^(a)Sequences from databases.bThese sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(c)These sequences were aligned to derive the corresponding primer.^(d)The nucleotide positions refer to the C. albicans tuf sequence fragment (SEQID NO. 2004).

TABLE 40 Specific and ubiquitous primers for nucleic acidamplification (atpD sequences). Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionBacterial species: Acinetobacter baumannii 1690 5′-CAG GTC CTG TTG CGA CTG AAG AA 243 186-208 1691^(b)5′-CAC AGA TAA ACC TGA GTG TGC TTT C 243 394-418Bacterial species: Bacteroides fragilis 2134  5′-CGC GTG AAG CTT CTG TG929 184-200 2135^(b) 5′-TCT CGC CGT TAT TCA GTT TC 929 395-414Bacterial species: Bordetella pertussis 2180  5′-TTC GCC GGC GTG GGC1672^(c) 544-558 2181^(b) 5′-AGC GCC ACG CGC AGG 1672^(c) 666-680Bacterial species: Enterococcus faecium 1698 5′-GGA ATC AAC AGA TGG TTT ACA AA 292 131-153 1699^(b)5′-GCA TCT TCT GGG AAA GGT GT 292 258-277 1700 5′-AAG ATG CGG AAA GAA GCG AA 292 271-290 1701^(b)5′-ATT ATG GAT CAG TTC TTG GAT CA 292 439-461Bacterial species: Klebsiella pneumoniae 1331 5′-GCC CTT GAG GTA CAG AAT GGT AAT GAA GTT 317  88-118 1332^(b)5′-GAC CGC GGC GCA GAC CAT CA 317 183-203^(a)These sequences were aligned to derive the corresponding primer.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(c)Sequence from databases. Bacterial species: Streptococcus agalactiae627 5′-ATT GTC TAT AAA AAT GGC GAT AAG TC 379-383^(a)  42-67^(b) 625^(c) 5′-CGT TGA AGA CAC GAC CCA AAG TAT CC 379-383^(a)  206-231^(b)628 5′-AAA ATG GCG ATA AGT CAC AAA AAG TA 379-383^(a)  52-77^(b) 625^(c) 5′-CGT TGA AGA CAC GAC CCA AAG TAT CC 379-383^(a)  206-231^(b)627 5′-ATT GTC TAT AAA AAT GGC GAT AAG TC 379-383^(a)  42-67^(b) 626^(c) 5′-TAC CAC CTT TTA AGT AAG GTG CTA AT 379-383^(a)  371-396^(b)628 5′-AAA ATG GCG ATA AGT CAC AAA AAG TA 379-383^(a)  52-77^(b) 626^(c) 5′-TAC CAC CTT TTA AGT AAG GTG CTA AT 379-383^(a)  371-396^(b)Bacterial group: Campylobacter jejuni and C. coli 2131 5′-AAG CMA TTG TTG TAA ATT TTG AAA G 1576, 1600,   7-31^(e)1849, 1863, 2139^(d,a) 2132^(c) 5′-TCA TAT CCA TAG CAA TAG TTC TA1576, 1600,   92-114^(e) 1849, 1863, 2139^(d,a)Bacterial genus: Bordetella sp. 8255′-ATG AGC ARC GSA ACC ATC GTT CAG TG 1672^(d)  1-26  826^(c)5′-TCG ATC GTG CCG ACC ATG TAG AAC GC 1672^(d) 1342-1367Fungal genus: Candida sp. 634 5′-AAC ACY GTC AGR RCI ATT GCY ATG GA460-472,  101-126^(f) 474-478^(a)  635^(c)5′-AAA CCR GTI ARR GCR ACT CTI GCT CT 460-472,  617-642^(f) 474-478^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the S. agalactiae atpD sequence fragment(SEQ ID NO. 380).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)Sequence from databases.^(e)The nucleotide positions refer to the C. jejuni atpD sequence fragment (SEQID NO. 1576).^(f)The nucleotide positions refer to the C. albicans atpD sequence fragment (SEQID NO. 460). Universal primers 5625′-CAR ATG RAY GAR CCI CCI GGI GYI MGI ATG 243, 244, 262,  528-557^(b)264, 280, 284, 291, 297, 309, 311, 315, 317, 324, 329, 332,334-336, 339, 342, 343, 351, 356, 357, 364-366, 370, 375, 379, 393^(a) 563^(c) 5′-GGY TGR TAI CCI ACI GCI GAI GGC AT 243, 244, 262, 687-712^(b) 264, 280, 284, 291, 297, 309, 311, 315, 317, 324, 329, 332,334-336, 339, 342, 343, 351, 356, 357, 364-366, 370, 375, 379, 393^(a)564 5′-TAY GGI CAR ATG AAY GAR CCI CCI GGI AA 243, 244, 262, 522-550^(b) 264, 280, 284, 291, 297, 309, 311, 315, 317, 324, 329, 332,334-336, 339, 342, 343, 351, 356, 357, 364-366, 370, 375, 379, 393^(a) 565^(c) 5′-GGY TGR TAI CCI ACI GCI GAI GGD AT 243, 244, 262, 687-712^(b) 264, 280, 284, 291, 297, 309, 311, 315, 317, 324, 329, 332,334-336, 339, 342, 343, 351, 356, 357, 364-366, 370, 375, 379, 393^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the K. pneumoniae atpD sequence fragment(SEQ ID NO. 317).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing. 6405′-TCC ATG GTI TWY GGI CAR ATG AA 248, 284, 315,  513-535^(b)317, 343, 357, 366, 370, 379, 393^(a)  641^(c)5′-TGA TAA CCW ACI GCI GAI GGC ATA CG 248, 284, 315,  684-709^(b)317, 343, 357, 366, 370, 379, 393^(a) 6425′-GGC GTI GGI GAR CGI ACI CGT GA 248, 284, 315,  438-460^(b)317, 343, 357, 366, 370, 379, 393^(a)  641^(c)5′-TGA TAA CCW ACI GCI GAI GGC ATA CG 248, 284, 315,  684-709^(b)317, 343, 357, 366, 370, 379, 393^(a) Sequencing primers 5665′-TTY GGI GGI GCI GGI GTI GGI AAR AC  669^(d) 445-470  567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG  669^(d) 883-908 5665′-TTY GGI GGI GCI GGI GTI GGI AAR AC  669^(d) 445-470 8145′-GCI GGC ACG TAC ACI GCC TG  666^(d) 901-920 5685′-RTI ATI GGI GCI GTI RTI GAY GT  669^(d) 25-47  567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG  669^(d) 883-908 5705′-RTI RYI GGI CCI GTI RTI GAY GT  672^(d) 31-53  567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG  669^(d) 883-908 5725′-RTI RTI GGI SCI GTI RTI GA  669^(d) 25-44  567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG  669^(d) 883-908 5695′-RTI RTI GGI SCI GTI RTI GAT AT  671^(d) 31-53  567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG  669^(d) 883-908 5715′-RTI RTI GGI CCI GTI RTI GAT GT  670^(d) 31-53  567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG 669^(d) 883-908^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the K. pneumoniae atpD sequence fragment(SEQ ID NO. 317).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)Sequences from databases. 700 5′-TIR TIG AYG TCG ART TCC CTC ARG 669^(a) 38-61  567^(b) 5′-TCR TCI GCI GGI ACR TAI AYI GCY TG  669^(a)883-908 568 5′-RTI ATI GGI GCI GTI RTI GAY GT  669^(a) 25-47  573^(b)5′-CCI CCI ACC ATR TAR AAI GC  666^(a) 1465-1484 5745′-ATI GCI ATG GAY GGI ACI GAR GG  666^(a) 283-305  573^(b)5′-CCI CCI ACC ATR TAR AAI GC  666^(a) 1465-1484 5745′-ATI GCI ATG GAY GGI ACI GAR GG  666^(a) 283-305  708^(b)5′-TCR TCC ATI CCI ARI ATI GCI ATI AT  666^(a) 1258-1283 6815′-GGI SSI TTY GGI ISI GGI AAR AC 685 694-716  682^(b)5′-GTI ACI GGY TCY TCR AAR TTI CCI CC 686 1177-1202 6815′-GGI SSI TTY GGI ISI GGI AAR AC 685 694-716  683^(b)5′-GTI ACI GGI TCI SWI AWR TCI CCI CC 685 1180-1205 6815′-GGI SSI TTY GGI ISI GGI AAR AC 685 694-716 6995′-GTI ACI GGY TCY TYR ARR TTI CCI CC 686 1177-1202 6815′-GGI SSI TTY GGI ISI GGI AAR AC 685 694-716  812^(b)5′-GTI ACI GGI TCY TYR ARR TTI CCI CC 685 1180-1205 1213 5′-AAR GGI GGI ACI GCI GCI ATH CCI GG  714^(a) 697-722 1212^(b)5′-CCI CCI RGI GGI GAI ACI GCW CC  714^(a) 1189-1211 1203 5′-GGI GAR MGI GGI AAY GAR ATG  709^(a) 724-744 1207^(b)5′-CCI TCI TCW CCI GGC ATY TC  709^(a)  985-1004 1204 5′-GCI AAY AAC ITC IWM YAT GCC  709^(a) 822-842 1206^(b)5′-CKI SRI GTI GAR TCI GCC A  709^(a) 926-944 1205 5′-AAY ACI TCI AWY ATG CCI GT  709^(a) 826-845 1207^(b)5′-CCI TCI TCW CCI GGC ATY TC  709^(a)  985-1004 2282 5′-AGR RGC IMA RAT GTA TGA  714^(a)  84-101 2284^(b)5′-TCT GWG TRA CIG GYT CKG AGA  714^(a) 1217-1237 2283 5′-ATI TAT GAY GGK ITT CAG AGG C  714^(a) 271-292 2285^(b)5′-CMC CIC CWG GTG GWG AWA C  714^(a) 1195-1213^(a)Sequences from databases.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.

TABLE 41 Internal hybridization probes for specific detectionof tuf sequences. Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Bacterial species: Abiotrophia adiacens2170  5′-ACG TGA CGT TGA CAA ACC A 1715  313-331 Bacterial species: Chlamydia pneumoniae 2089 5′-ATG CTG AAC TTA TTG ACC TT  20 136-155  2090 5′-CGT TAC TGG AGT CGA AAT G  20 467-485 Bacterial species: Enterococcus faecalis 5805′-GCT AAA CCA GCT ACA ATC ACT CCA C 62-63, 607^(a) 584-608^(b) 6035′-GGT ATT AAA GAC GAA ACA TC 62-63,  607^(a) 440-459^(b) 1174 5′-GAA CGT GGT GAA GTT CGC 62-63, 607^(a) 398-415^(b)Bacterial species: Enterococcus faecium 6025′-AAG TTG AAG TTG TTG GTA TT 64, 608^(a) 426-445^(c)Bacterial species: Enterococcus gallinarum 6045′-GGT GAT GAA GTA GAA ATC GT 66, 609^(a) 419-438^(d)Bacterial species: Escherichia coli 579 5′-GAA GGC CGT GCT GGT GAG AA 78 503-522  2168  5′-CAT CAA AGT TGG TGA AGA AGT TG  78 409-431 Bacterial species: Neisseria gonorrhoeae 2166 5′-GAC AAA CCA TTC CTG CTG 126 322-339^(e)Fungal species: Candida albicans 577 5′-CAT GAT TGA ACC ATC CAC CA407-411^(a) 406-425^(f) Fungal species: Candida dubliniensis 5785′-CAT GAT TGA AGC TTC CAC CA 412, 414-415^(a) 418-437^(g)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the E. faecalis tuf sequence fragment (SEQID NO. 607).^(c)The nucleotide positions refer to the E. faecium tuf sequence fragment (SEQID NO. 608).^(d)The nucleotide positions refer to the E. gallinarum tuf sequence fragment(SEQ ID NO. 609).^(e)The nucleotide positions refer to the N. gonorrhoeae tuf sequence fragment(SEQ ID NO. 126).^(f)The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment(SEQ ID NO. 408).^(g)The nucleotide positions refer to the C. dubliniensis tuf(EF-1) sequencefragment (SEQ ID NO. 414). Bacterial species: Haemophilus influenzae 5815′-ACA TCG GTG CAT TAT TAC GTG G  610^(a) 551-572 Bacterial species: Mycoplasma pneumoniae 2095 5′-CGG TCG GGT TGA ACG TGG2097^(a) 687-704  Bacterial species: Staphylococcus aureus 5845′-ACA TGA CAC ATC TAA AAC AA 176-180^(b) 369-388^(c) 5855′-ACC ACA TAC TGA ATT CAA AG 176-180^(b) 525-544^(c) 5865′-CAG AAG TAT ACG TAT TAT CA 176-180^(b) 545-564^(c) 5875′-CGT ATT ATC AAA AGA CGA AG 176-180^(b) 555-574^(c) 5885′-TCT TCT CAA ACT ATC GTC CA 176-180^(b) 593-612^(c)Bacterial species: Staphylococcus epidermidis 5895′-GCA CGA AAC TTC TAA AAC AA 185, 611^(b) 445-464^(d) 5905′-TAT ACG TAT TAT CTA AAG AT 185, 611^(b) 627-646^(d) 5915′-TCC TGG TTC TAT TAC ACC AC 185, 611^(b) 586-605^(d) 5925′-CAA AGC TGA AGT ATA CGT AT 185, 611^(b) 616-635^(d) 5935′-TTC ACT AAC TAT CGC CCA CA 185, 611^(b) 671-690^(d)Bacterial species: Staphylococcus haemolyticus 5945′-ATT GGT ATC CAT GAC ACT TC 186, 188-190^(b) 437-456^(e) 5955′-TTA AAG CAG ACG TAT ACG TT 186, 188-190^(b) 615-634^(e)Bacterial species: Staphylococcus hominis 5965′-GAA ATT ATT GGT ATC AAA GA 191, 193-196^(b) 431-450^(f) 5975′-ATT GGT ATC AAA GAA ACT TC 191, 193-196^(b) 437-456^(f) 5985′-AAT TAC ACC TCA CAC AAA AT 191, 193-196^(b) 595-614^(f)^(a)Sequences from databases.^(b)These sequences were aligned to derive the corresponding probe.^(c)The nucleotide positions refer to the S. aureus tuf sequence fragment (SEQ IDNO. 179).^(d)The nucleotide positions refer to the S. epidermidis tuf sequence fragment(SEQ ID NO. 611).^(e)The nucleotide positions refer to the S. haemolyticus tuf sequence fragment(SEQ ID NO. 186).^(f)The nucleotide positions refer to the S. hominis tuf sequence fragment (SEQID NO. 191). Bacterial species: Staphylococcus saprophyticus 5995′-CGG TGA AGA AAT CGA AAT CA 198-200^(a) 406-425^(b) 6005′-ATG CAA GAA GAA TCA AGC AA 198-200^(a) 431-450^(b) 6015′-GTT TCA CGT GAT GAT GTA CA 198-200^(a) 536-555^(b) 6955′-GTT TCA CGT GAT GAC GTA CA 198-200^(a) 563-582^(b)Bacterial species: Streptococcus agalactiae 582^(c)5′-TTT CAA CTT CGT CGT TGA CAC GAA CAG T 207-210^(a) 404-431^(d) 583^(c)5′-CAA CTG CTT TTT GGA TAT CTT CTT TAA TAC CAA CG 207-210^(a)433-467^(d) 1199  5′-GTA TTA AAG AAG ATA TCC AAA AAG C 207-210^(a)438-462^(d) Bacterial species: Streptococcus pneumoniae 1201 5′-TCA AAG AAG AAA CTA AAA AAG CTG T 971, 977, 513-537^(e) 979, 986^(a)Bacterial species: Streptococcus pyogenes 1200 5′-TCA AAG AAG AAA CTA AAA AAG CTG T 1002  473-497 Bacterial group: Enterococcus casseliflavus-flavescens- gallinarum group620 5′-ATT GGT GCA TTG CTA CGT 58, 65, 66^(a) 527-544^(f) 1122 5′-TGG TGC ATT GCT ACG TGG 58, 65, 66^(a) 529-546^(f)Bacterial group: Enterococcus sp., Gemella sp., A. adiacens 2172 5′-GTG TTG AAA TGT TCC GTA AA 58-62, 67-71, 477-496^(g) 87-88, 607-609,727, 871 1715, 1722^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the S. saprophyticus tuf sequence fragment(SEQ ID NO. 198).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the S. agalactiae tuf sequence fragment(SEQ ID NO. 209).^(e)The nucleotide positions refer to the S. pneumoniae tuf sequence fragment(SEQ ID NO. 986).^(f)The nucleotide positions refer to the E. flavescens tuf sequence fragment(SEQ ID NO. 65).^(g)The nucleotide positions refer to the E. faecium tuf sequence fragment (SEQID NO. 608). Bacterial genus: Gemella 2171 5′-TCG TTG GAT TAA CTG AAG AA 87, 88^(a) 430-449^(b)Bacterial genus: Staphylococcus sp. 605 5′-GAA ATG TTC CGT AAA TTA TT176-203^(a) 403-422^(c) 606 5′-ATT AGA CTA CGC TGA AGC TG 176-203^(a)420-439^(c) 1175  5′-GTT ACT GGT GTA GAA ATG TTC 176-203^(a) 391-411^(c)1176  5′-TAC TGG TGT AGA AAT GTT C 176-203^(a) 393-411^(c)Bacterial genus: Streptococcus sp. 1202 5′-GTG TTG AAA TGT TCC GTA AAC A 206-231, 971, 466-487^(d)977, 979, 982-986^(a) Fungal species: Candida albicans 1156 5′-GTT GAA ATG CAT CAC GAA CAA TT 407-412, 624^(a) 680-702^(e)Fungal group: Candida albicans and C. tropicalis 1160 5′-CGT TTC TGT TAA AGA AAT TAG AAG 407-412, 748-771^(e) 429, 624^(a)Fungal species: Candida dubliniensis 1166 5′-ACG TTA AGA ATG TTT CTG TCA A 414-415^(a) 750-771^(f) 1168 5′-GAA CAA TTG GTT GAA GGT GT 414-415^(a) 707-726^(f)Fungal species: Candida glabrata 1158  5′-AAG AGG TAA TGT CTG TGG T 417781-799  1159  5′-TGA AGG TTT GCC AGG TGA 417 718-735 Fungal species: Candida krusei 1161  5′-TCC AGG TGA TAA CGT TGG 422720-737 ^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the G. haemolysans tuf sequence fragment(SEQ ID NO. 87).^(c)The nucleotide positions refer to the S. aureus tuf sequence fragment (SEQ IDNO. 179).^(d)The nucleotide positions refer to the S. pneumoniae tuf sequence fragment(SEQ ID NO. 986).^(e)The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment(SEQ ID NO. 408).^(f)The nucleotide positions refer to the C. dubliniensis tuf(EF-1) sequencefragment (SEQ ID NO. 414).Fungal group: Candida lusitaniae and C. guillermondii 1162 5′-CAA GTC CGT GGA AAT GCA 418, 424^(a) 682-699^(b)Fungal species: Candida parapsilosis 1157 5′-AAG AAC GTT TCA GTT AAG GAA AT 426 749-771 Fungal species: Candida zeylanoides 1165  5′-GGT TTC AAC GTG AAG AAC 432713-730  Fungal genus: Candida sp. 1163  5′-GTT GGT TTC AAC GTT AAG AAC407-412, 414-415, 728-748^(c) 417, 418, 422, 429^(a) 1164 5′-GGT TTC AAC GTC AAG AAC 413, 416, 420, 740-757^(b) 421, 424, 425,426, 428, 431^(a) 1167  5′-GTT GGT TTC AAC GT 406-426, 428-432,728-741^(c) 624^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the C. lusitaniae tuf(EF-1) sequencefragment (SEQ ID NO. 424).^(c)The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment(SEQ ID NO. 408).

TABLE 42Strategy for the selection of amplification/sequencing primers from atpD (F-type) sequences.SEQ ID23                         49   443                           472   881                           910NO.: Accession #: B. cepaciaAGTgCAT CGGCGCCGTT ATCGACGTGG...TGTTCG GCGGTGCTGG CGTGGGCAAG ACCG...TCCA GGCCGTGT ACGTCCCTGC GGACGACT2728 X76877 B. pertussisAGTgCAT CGGCGCCGTG GTGGATATTC...TGTTCG GCGGCGCCGG CGTGGGCAAG ACCG...TCCA GGCCGTGT ACGTGCCTGC CGACGACT2729 Genome project P. aeruginosaAAATCAT CGGCGCCGTG ATCGACGTGG...TGTTCG GCGGCGCCGG CGTGGGCAAG ACCG...TCCA GGCCGTAT ACGTTCCCGC GGACGACC2730 Genome project E. coliAGGTAAT CGGCGCCGTA GTTGACGTCG...TGTTCG GTGGTGCGGG TGTAGGTAAA ACCG...TACA GGCAGTAT ACGTACCTGC GGATGACT2731 J01594 N. gonorrhoeaeAAATTAT CGGTGCGGTT GTTGACGTGG...TGTTCG GCGGTGCCGG TGTGGGTAAA ACCG...TCCA AGCCGTAT ATGTACCTGC GGATGACT2732 Genome project M. thermoaceticaAGGTTAT TGGCCCGGTG GTTGACGTCG...TCTTCG GCGGCGCCGG GGTCGGCAAG ACGG...TGCA AGCTATCT ATGTGCCGGC CGACGACC2733 U64318 S. aurantiacaAGGTTcT CGGTCCCGTG ATTGACGTGG...TGTTCG GCGGCGCCGG CGTGGGCAAG ACGG...TGCA GGCCATCT ACGTGCCCGC CGACGACC2734 X76879 M. tuberculosisGGGTCAC TGGGCCCGTC GTCGACGTCG...TGTTCG GCGGTGCCGG GGTGGGCAAG ACGG...TGCA AGCCGTCT ACGTGCCCGC CGACGACT2735 Z73419 B. fragilisAGGTAAT TGGCCCTGTG GTCGATGTGT...TGTTTG GCGGGGCCGG AGTGGGTAAA ACTG...TGCA GGCTGTTT ACGTACCGGC TGATGACT2736 M22247 C. lyticaAAATTAT TGGCCCAGTT ATAGATGTGG...TATTTG GAGGTGCCGG AGTAGGTAAA ACAG...TACA GGCGGTTT ACGTACCTGC GGATGATT672 M22535 A. woodiiAGGTTAT TGGACCAGTA GTCGATGTTA...TTTTCG GTGGTGCCGG AGTTGGTAAA ACCG...TTCA GGCCGTTT ACGaTCCAGC CGATGACT2737 U10505 C. acetobutylicumAGGTAAT AGGACCTGTT GTGGATATTA...TGTTCG GTGGTGCCGG TGTTGGTAAA ACAG...TTCA GGCTGTAT ATGTTCCTGC TGATGACC671 AF101055 M. pneumoniaeAAGTGAT TGGCCCGGTA GTTGATGTCA...TATTTG GTGGTGCTGG TGTTGGTAAA ACGG...TGCA AGCGATCT ATGTGCCAGC TGATGACT2738 U43738 H. pyloriAGGTTtT AGGCCCGGTG GTAGATGTGG...TGTTTG GTGGGGCTGG CGTAGGCAAA ACGG...TTCA AGCGGTGT ATGTGCCAGC AGACGACT670 AF004014 Selected sequences   RTIAT IGGIGCIGTI RTIGAYGT 568for universal primers   RTIRY IGGICCIGTI RTIGAYGT 570  RTIRT IGGISCIGTI RTIGA 572   RTIRT IGGISCIGTI RTIGATAT 569  RTIRT IGGICCIGTI RTIGATGT 571                                  TTYG GIGGIGCIGG IGTIGGIAAR AC 566Selected sequence                                                                      CA RGCIRTIT AYGTICCIGC IGAYGA567 for universal primer^(a) The sequence numbering refers to theEscherichia coli atpD gene fragment (SEQ ID NO. 669). Nucleotides incapitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a)This sequence is the reverse-complement of the selectedprimer.

TABLE 43Strategy for the selection of universal amplification/sequencing primersfrom atpD (V-type) sequences.691                          719   1177                           1208SEQ ID NO.: E. hiraeCC AGGTCCGTTT GGTGCAGGGA AGACAGT...TCTGGTGGAg ATaTCtctGA ACCAGTGACT CA685 H. salinarumCC GGGGCCGTTC GGGTCCGGGA AGACGGT...CCCGGCGGGg ACTTCtccGA GCCGGTCACC CA687 T. thermophilusCC TGGGCCCTTC GGCAGCGGCA AGACCGT...CCGGGCGGCg ACaTgtccGA GCCCGTGACC CA693 HumanCC TGGGGCCTTC GGATGTGGCA AGACTGT...CCCGGTGGAg ACTTCtcAGA tCCCGTGACG AC688 T. congolenseCC TGGCGCGTTT GGATGCGGAA AGACGGT...CCTGGAGGTg ACTTTtctGA cCCAGTGACG TC692 P. falciparumCC TGGTGCATTT GGTTGTGGAA AAACTTG...CCAGGTGGTg ATTTCtctGA cCCTGTAACT AC689 C. pneumoniae CC AGGACCTTTT GGTGCAGGGA AAACAGT...GCAGGAGGA A  ACTTTGA AGA ACCAGTCACT CA 686 Selected sequences    GGISSITTY GGIISIGGIA ARAC 681 for universal primersSelected sequences                                      GGIGGIA AYTTYGARGA RCCIGTIAC 682for universal                                      GGIGGIG AYWTIWSIGA ICCIGTIAC 683primers^(a) The sequence numbering refers to the Enterococcus hirae atpDgene fragment (SEQ ID NO. 685). Nucleotides in capitals are identical tothe selected sequences or match those sequences. Mismatches for SEQ IDNOs. 681 and 682 are indicated by lower-case letters. Mismatches for SEQID NO. 683 are indicated by underlined nucleotides. Dots indicate gapsin the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a)These sequences are the reverse-complement of theselected primers.

TABLE 44Strategy for the selection of universal amplification/sequencing primersfrom tuf (M) sequences (organelle origin).601                                635   1479                            1511SEQ ID NO.: Accession #: C. neoformans ^(a)AAGAA CATGATCACC GGTaCCtCCC AGgctGACTG...CGCc g TCcG A  GA cat G c G AC  A G A CcGTTGc CGT 2739 U81803 S. cerevisiae ^(a)AAGAA CATGATTACT GGTaCTtCTC AAgctGACTG...CGCT g TC A G A  GA catGa G AC  A A A CTGTcGc TGT 665 X00779 O. volvulus ^(a)AAGAA TATGATCACA GGTaCTtCTC AGgctGACTG...TGCT g TGcGt GA tatGa G A C  AA A CaGTTGc GGT 2740 M64333 Human^(a)AAAAA CATGATTACA GGGaCAtCTC AGgctGACTG...TGCT g TTcGt GA tatGa G A C  AG A CaGTTGc TGT 2741 X03558 G. max B1^(b)AAGAA CATGATCACC GGCGCTGCCC AGATGGACGG...TGCTAT TA G A  GA A GG A GGC A  A A A CTGTTGG AGC 2742 Y15107 G. max B2^(b)AAAAA CATGATCACC GGCGCCGCCC AGATGGACGG...TGCTAT TA G A  GA A GG A GGC A  A A A CTGTTGG AGC 2743 Y15108 E. coli ^(c)AAAAA CATGATCACC GGTGCTGCTC AGATGGACGG...CGCaATCcGt GA A GGCGGCC GT ACcGTTGG CGC 78 — S. aureofaciens ^(c)AAGAA CATGATCACC GGTGCCGCCC AGATGGACGG...CGCcATCcGt GAGGGTGG T C GT ACcGTgGG CGC 2744 AF007125 E. tenella ^(b)AAAAA TATGATTACA GGAGCAGCAC AAATGGATGG...TGCTAT AA G A  GA A GG A GG AA  A A A CT A TAGG AGC 2745 AI755521 T. gondii ^(b)AAGAA TATGATTACT GGAGCCGCAC AAATGGATGG...TGCTAT TA G A  GA A GG A GG TC GT A CT A TAGG AGC 2746 Y11431 S. cerevisiae ^(b)AAGAA TATGATTACC GGTGCTGCTC AAATGGATGG...CAATATC A G A  GAGGGTGG AA  GAA CTGTTGG TAC 619 K00428 A. thaliana ^(b)AAAAA TATGATTACT GGAGCTGCGC AAATGGATGG...TGCc t T AA G G  GA A GG A GGTA  GA A CaGTTGG AGC 2747 X89227 Selected sequence for universal primer   AA YATGATIACI GGIGCIGCIC ARATGGA 664Selected sequences for universal primers                                              TATIAGR GARGGIGGIM RIACTRTWGG^(d) 652                                             ATCCGT GAGGGYGGCC GITCIGT^(d) 561 The sequence numbering refers to the Saccharomyces cerevisiaetuf (M) gene (SEQ ID NO. 619). Nucleotides in capitals are identical tothe selected sequences or match those sequences. Mismatches for SEQ IDNOs. 652 and 664 are indicated by lower-case letters. Mismatches for SEQID NO. 561 are indicated by underlined nucleotides. Dots indicate gapsin the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a)This sequence refers to tuf (EF-1) gene. ^(b)Thissequence refers to tuf (M) or organelle gene. ^(c)This sequence refersto tuf gene from bacteria. ^(d)These sequences are thereverse-complement of the selected primers.

TABLE 45Strategy for the selection of eukaryotic sequencing primers from tuf (EF-1) sequences.154                       179   286                          314SEQ ID NO.: Accession #: S. cerevisiaeGG TTCTTTCAAG TACGCTTGGG TTTT...AGAGA TTTCATCAAG AACATGATTA CTGG... 665X00779 B. hominisGG CTCCTTCAAG TACGCGTGGG TGCT...CGTGA CTTCATaAAG AACATGATCA CGGG... 2748D64080 C. albicansGG TTCTTTCAAA TACGCTTGGG TCTT...AGAGA TTTCATCAAG AATATGATCA CTGG... 2749M29934 C. neoformansTC TTCTTTCAAG TACGCTTGGG TTCT...CGAGA CTTCATCAAG AACATGATCA CCGG... 2739U81803 E. histolyticaGG ATCATTCAAA TATGCTTGGG TCTT...AGAGA TTTCATTAAG AACATGATTA CTGG... 2751M92073 G. lambliaGG CTCCTTCAAG TACGCGTGGG TCCT...CGCGA CTTCATCAAG AACATGATCA CGGG... 2752D14342 H. capsulatumAA ATCCTTCAAA TATGCGTGGG TCCT...CGTGA CTTCATCAAG AACATGATCA CTGG... 2753U14100 Human GG CTCCTTCAAG TATGCCTGGG TCTT...AGAGA CTT tATCAAA AACATGATTA CAGG... 2741 X03558 L. braziliensisGC GTCCTTCAAG TACGCGTGGG TGCT...CGCGA CTTCATCAAG AACATGATCA CCGG... 2755U72244 O. volvulusGG CTCATTTAAA TATGCTTGGG TATT...CGTGA TTTCATTAAG AATATGATCA CAGG... 2740M64333 P. berghei GG TagTTTCAAA TATGCATGGG TTTT...AAA c A TTT tATTAAA AATATGATTA CTGG... 2757 AJ224150 P. knowlesiGG AagTTTTAAG TACGCATGGG TGTT...AAGGA TTTCATTAAA AATATGATTA CCGG... 2758AJ224153 S. pombeGG TTCCTTCAAG TACGCCTGGG TTTT...CGTGA TTTCATCAAG AACATGATTA CCGG... 2759U42189 T. cruziTC TTCTTTCAAG TACGCGTGGG TCTT...CGCGA CTTCATCAAG AACATGATCA CGGG... 2760L76077 Y. lipolyticaGG TTCTTTCAAG TACGCTTGGG TTCT...CGAGA TTTCATCAAG AACATGATCA CCGG... 2761AF054510 Selected sequences for amplification primers    TCITTYAAR TAYGCITGGG T 558                                   GA YTTCATYAAR AAYATGATYA C 560                                   GA YTTCATIAAR AAYATGAT 653The sequence numbering refers to the Saccharomyces cerevisiae tuf (EF-1) gene fragment (SEQ ID NO. 665). Nucleotides incapitals are identical to the selected sequences SEQ ID NOs. 558, 560 or 653, or match those sequences. Mismatches forSEQ ID no. 558 and 560 are indicated by lower-case letters. Mismatches for SEQ ID NO. 653 are indicated by underlinednucleotides. Dots indicate gaps in the sequences displayed. “R” “Y” “M”“K” “W” and “S”designate nucleotide positions which are degenerated. “R”stands for A or G; “Y” stands for C or T; “M” stands for A or C; “K”stands for G or T; “W” stands for A or T; “S” stands for C or G. “I”stands for inosinewhich is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.   751                      776   1276                        1304SEQ ID NO.: Accession #: S. cerevisiae...GTTTACAA GATCGGTGGT ATTGGTAC...GACATG AGACAAACTG TCGCTGTCGG TGT 665X00779 B. hominis...GTGTACAA GATTGGCGGT ATTGGTAC...GATATG AGACAGACTG TCGCTGTCGG TAT 2748D64080 C. albicans...GTTTACAA GATCGGTGGT ATTGGTAC...GATATG AGACAAACCG TTGCTGTtGG TGT 2749M29934 C. neoformans...GTCTACAA GATCGGTGGT ATCGGCAC...GACATG CGACAGACCG TTGCCGTtGG TGT 2750U81803 E. histolytica...GTTTACAA GATTTcAGGT ATTGGAAC...GATATG AaACAAACCG TTGCTGTtGG AGT 2751M92073 G. lamblia...GTCTACAA GATCTcGGGc gTCGGGAC...~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~ 2752D14342 H. capsulatum...GTGTACAA AATCTcTGGT ATTGGCAC...GACATG AGACAAACCG TCGCTGTCGG TGT 2753U14100 Human...GTCTACAA AATTGGTGGT ATTGGTAC...GATATG AGACAGACAG TTGCgGTgGG TGT 2754X03558 L. braziliensis...GTGTACAA GATCGGCGGT ATCGGCAC...GACATG CGCagAACGG TCGCCGTCGG CAT 2755U72244 O. volvulus...GTTTACAA AATTGGAGGT ATTGGAAC...GATATG AGACAAACAG TTGCTGTtGG CGT 2756M64333 P. berghei...GTATACAA AATTGGTGGT ATTGGTAC...GATATG AGACAAACAA TTGCTGTCGG TAT 2757AJ224150 P. knowlesi...GTATACAA AATCGGTGGT ATTGGTAC...GATATG AGACAAACCA TTGCTGTCGG TAT 2758AJ224153 S. pombe...GTTTACAA GATCGGTGGT ATTGGTAC...GACATG CGTCAAACCG TCGCTGTCGG TGT 2759U42189 T. cruzi...GTGTACAA GATCGGCGGT ATCGGCAC...GACATG CGCCAGACGG TCGCCGTCGG CAT 2760L76077 Y. lipolytica...GTCTACAA GATCGGTGGT ATCGGCAC...GACATG CGACAGACCG TTGCTGTCGG TGT 2761AF054510 Selected sequence for amplification primer      TACAA RATYKGIGGT ATYGG 654Selected sequences for amplification primers^(a)      TACAA RATYKGIGGT ATYGG 655                                     ATG MGICARACIR TYGCYGTCGG 559The sequence numbering refers to the Saccharomyces cerevisiae tuf (EF-1) gene fragment (SEQ ID NO. 665). Nucleotides incapitals are identical to the selected sequences or match those sequences. Mismatches are indicated by lower-caseletters. “~”indicate incomplete sequence data. Dots indicate gaps in the sequences displayed.“R” “Y” “M” “K” “W” and “S”designate nucleotide positions which are degenerated. “R”stands for A or G; “Y” stands for C or T; “M” stands for A or C; “K”stands for G or T; “W” stands for A or T; “S” stands for C or G. “I”stands for inosinewhich is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.^(a)This sequences are the reverse-complement of the selected primers.

TABLE 46 Strategy for the selection of Streptococcus agalactiae-specificamplification primers from tuf sequences. SEQ ID Accession305                           334   517                       542 NO.:#: S. agalactiaeCCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG 207 —S. agalactiaeCCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG 208 —S. agalactiaeCCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG 209 —S. agalactiaeCCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG 210 —S. anginosusCCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AACGTaGGgG TTCTTCTTCG TG 211 —S. anginosusCCAGAA CGTGATACTG ACAAACCaTT gCTT...AGAt AACGTaGGgG TTCTTCTTCG TG 221 —S. bovisCCAaAA CGTGATACTG ACAAACCaTT gCTT...GGAt AACGTTGGTG TTCTTCTTCG TG 212 —S. gordoniiCCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AAtGTaGGTG TcCTTCTTCG TG 223 —S. mutansCCAGAA CGTGATACTG ACAAgCCgcT cCTT...GGAt AAtGTTGGTG TTCTcCTTCG TG 224 —S. pneumoniaeCCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AACGTaGGTG TcCTTCTTCG TG2861^(c) S. sanguinisCCAGAA CGcGATACTG ACAAgCCaTT gCTT...GGAC AACGTaGGTG TgCTTCTcCG TG 227 —S. sobrinusCCAaAA CGcGATACTG AtAAgCCaTT gCTT...AGAt AACGTTGGTG TgCTTCTTCG TG 228 —B. cepaciaCCGGAg CGTGcagtTG ACggcgCgTT cCTG...CGAC AACGTTGGTa TcCTgCTgCG cG 16 —B. fragilisCCTccg CGcGATgtTG AtAAACCTTT ctTG...TGAC AACGTaGGTc TgtTgCTTCG TG 2762P33165 B. subtilisCCAGAA CGcGAcACTG AaAAACCaTT caTG...TGAC AACaTTGGTG ccCTTCTTCG cG 2763Z99104 C. diphtheriaeCCAGAg CGTGAgACcG ACAAgCCaTT cCTC...CGAC AACtgTGGTc TgCTTCTcCG TG 662 —C. trachomatisCCAGAA aGaGAaAtTG ACAAgCCTTT cTTA...AGAg AAtGTTGGat TgCTcCTcaG aG 22 —E. coliCCAGAg CGTGcgAtTG ACAAgCCgTT cCTg...TGAg AACGTaGGTG TTCTgCTgCG TG 78 —G. vaginalisCCAact CacGATctTG ACAAgCCaTT cTTg...CGAt RACacTGGTc TTCTTCTcCG cG2856^(a) S. aureusCCAGAA CGTGATtCTG ACAAACCaTT cATg...TGAC AACaTTGGTG catTatTaCG TG 179 —Selected sequence for    GAA CGTGATACTG ACAAACCTTT A 549species-specific primer Selected sequence for                                       C AACGTTGGTG TTCTTCTTC 550species-specific primer^(b) The sequence numbering refers to theStreptococcus agalactiae tuf gene fragment (SEQ ID NO. 209). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a)This sequence corresponds to SEQ ID No. 135 of patentpublication WO98/20157. ^(b)This sequence is the reverse-complement ofthe selected primer. ^(c)This sequence corresponds to SEQ ID NO. 145 ofpatent publication WO98/20157.

TABLE 47 Strategy for the selection of Streptococcus agalactiae-specifichybridization probes from tuf sequences. SEQ Acces- ID sion401                            431 433                                    470NO.: #: S.GGTACTGT TaaaGTtAAt GACGAAGTTG AAATCGTTGG TATcAAAGAc GAaATCtctA AAGCAGTTGT TA206 acidominimus S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA209 S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA2860^(a) S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA207 S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA210 S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA208 S. anginosusGGTACTGT TaaaGTCAAC GACGAAGTTG AAATCGTTGG TATccgtGAt GAaATCCAAA AAGCAGTTGT TA211 S. anginosusGGTACTGT TaaaGTCAAC GAtGAAGTTG AAATCGTTGG TATccgcGAg GAaATCCAAA AAGCAGTTGT TA221 S. bovisGGTACTGT TaaaGTCAAC GACGAAGTTG AAATCGTTGG TATccgtGAc GAcATCCAAA AAGCtGTTGT TA212 S. anginosusGGTACTGT TaaaGTCAAt GAtGAAGTTG AAATtGTTGG TATTcgtGAc GAaATCCAAA AAGCAGTTGT TA213 S. cricetusGGTACTGT TaagGTCAAt GACGAAGTTG AAATCGTTGG TATcAAgGAc GAaATCCAAA AAGCgGTTGT TA214 S. cristatusGGTACTGT TCGTGTCAAC GAtGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA215 S. downeiGGTACTGT TaagGTCAAC GACGAAGTTG AAATCGTTGG TATcAAgGAc GAaATCCAAA AAGCAGTTGT TA216 S.GGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActaAAA AAGCtGTTGT TA217 dysgalactiae S. equi equiGGTACTGT TCGTGTtAAC GACGAAaTcG AAATCGTTGG TATcAgAGAc GAgATCaAAA AAGCAGTTGT TA218 S. ferusGGTACTGT aaGaGTCAAC GAtGAAGTTG AAATCGTTGG TATcAAAGAc GAaATCactA AAGCAGTTGT TA219 S. gordoniiGGTAtcGT TaaaGTCAAt GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA220 S. macacaeGGTACTGT TaagGTtAAt GAtGAAGTTG AAATCGTTGG TATTcgtGAc GATATtCAAA AAGCAGTTGT TA222 S. gordoniiGGTAtcGT TaaaGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActCAAA AAGCAGTTGT TA223 S. mutansGGTACTGT TaaaGTtAAC GAtGAAGTTG AAATCGTTGG TATccgtGAt GAcATtCAAA AAGCtGTTGT TA224 S. oralisGGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActCAAA AAGCAGTTGT TA2764 P33170 S.GGTgtTGT TCGTGTCAAt GAtGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA225 parasanguinis S. pneumoniaeGGTAtcGT TaaaGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActCAAA AAGCAGTTGT TA2861^(c) S. pyogenesGGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActaAAA AAGCtGTTGT TA2765 Genome proj- ect S. rattiGGTACTGT TaaaGTCAAt GACGAAGTTG AAATCGTTGG TATccgtGAt GAcATCCAAA AAGCtGTTGT TA226 S. salivariusGGTgtTGT TCGTGTCAAt GACGAAGTTG AAATCGTTGG TcTTAAAGAA GAcATCCAAA AAGCAGTTGT TA2862^(d) S. sanguinisGGTAtcGT TaaaGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA227 S. sobrinusGGTACTGT TaagGTtAAC GACGAAGTTG AAATCGTTGG TATccgtGAc GATATCCAAA AAGCAGTTGT TA228 S. suisGGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TcTTcAAGAA GAaAaatctA AAGCAGTTGT TA229 S. uberisGGTACTGT TCGTGTCAAC GACGAAaTTG AAATCGTTGG TATcAAAGAA GAaActaAAA AAGCAGTTGT TA230 S.GGTgtTGT TCGTGTtAAt GACGAAGTTG AAATCGTTGG TcTTAAAGAA GAaATCCAAA AAGCAGTTGT TA231 vestibularis Selected    ACTGT TCGTGTCAAC GACGAAGTTG AAA 582sequences for                                   CGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTG583 species- specific hybridization probes^(b) The sequence numberingrefers to the Streptococcus agalactiae tuf gene fragment (SEQ ID NO.209). Nucleotides in capitals are identical to the selected sequences ormatch those sequences. Mismatches are indicated by lower-case letters.Dots indicate gaps in the sequences displayed. ^(a)This sequencecorresponds to SEQ ID No. 144 of patent publication WO98/20157.^(b)These sequences are the reverse-complement of the selected probes.^(c)This sequence corresponds to SEQ ID NO. 145 of patent publicationWO98/20157. ^(d)This sequence corresponds to SEQ ID NO. 146 of patentpublication WO98/20157.

TABLE 48 Strategy for the selection of Streptococcus agalactiae-specificamplification primers from atpD sequences. SEQ ID39                                         80   203                             234   368                             399NO.: S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG380 S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG379 S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG381 S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG382 S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG383 S. bovisTT GATTGTtTAT AAAgATGGCG ATAAGTCtCA AAAAaTcGTg...TAAaGAaA CTTTGGGTCG TGTgTTtAAt GTTC...CcT tcTtGCcCCT TACcTAAAAG GTGGTAAAG2766 S. salivariusTT GgTcGTtTAT ActgATGaac AaAAGTCtaA AcgtaTcGTg...TAAaGATA CccTtGGaCG TGTCTTtAAC GTTC...CTT gcTAGCcCCT TACcTtAAgG GTGGTAAAG387 S. pneumoniaecT tgTcGTCTAc AAAAATGaCG AaAgaaaAac AAAAaTcGTc...TAAaGAaA CTTTGGGaCG TGTCTTCAAC GTTt...CcT tcTtGCcCCT TACcTtAAAG GTGGTAAAG2767 S. pyogenesTT GATTGTtTAT AAAgATaGtG ATAAaaagCA AAAAaTcGTc...TAAaGAaA CTTTGGGaCG cGTCTTtAAt GTaC...CcT tcTtGCcCCT TACcTtAAAG GTGGTAAAG2768 S. anginosuscT tgTaGTCTAT AAAAATGaCG AaAAtaaAtc AAAAaTcGTc...gAAaGAaA CacTtGGTCG cGTCTTtAAC GTTt...CcT tTTAGCcCCc TACcTcAAAG GTGGgAAAG386 S. sanguiniscT tgTaGTCTAT AAAAATGatG AgAAaaaAtc AAAAaTcGTc...aAAGGAaA CTcTaGGcCG gGTgTTCAAt GTTt...CcT gcTAGCACCT TAtcTgAAAG GTGGgAAAG2769 S. mutansTT GgTcGTtTAT AAAgATGGCG AcAAGTCtCA AAgAaTtGTt...aAAaGAaA CacTaGGTCG TGTCTTtAAt GTTC...CcT tcTtGCcCCT TAtcTtAAAG GTGGTAAAG2770 B. anthracisgT aAaacagagc AAcgAaaaCG gaAcaagcat tAActTAacA...TgAtGcaA CacTtGGTCG TGTaTTtAAC GTat...CTT AcTtGCtCCT TACaTtAAgG GTGGTAAga247 B. cereusgT aAaacaaagc AAcgAaaaCG g...aagcat gAActTAacA...TgAtGcaA CacTtGGaCG TGTaTTCAAC GTat...CTT AcTtGCtCCT TACaTtAAgG GTGGTAAga248 E. faeciumTT agTTGTtTAT AAAAATGaCG AaAAtaaAtc AAAAGTtGTt...TAAaGAaA CaTTaGGTCG cGTaTTCAAC GTaC...tTT gcTtGCcCCa TAtTTAAAAG GTGGgAAAG292 E. gallinarumTT GATcGTtTAc AAAAAaGaCG AgAAaaaAac AAAAGTAGTA...aAcaGATA CTcTaGGcCG aGTaTTtAAt GTaC...tTT ATTAGCtCCT TACTTAAAAG GTGGTAAAG293 E. faecalisTT agTcGTtTAT AAAAATGGCG AagcaaaACA AAAAGTAGTA...TAAaGATA CaTTaGGTCG TGTgTTtAAC GTTt...CTT ATTAGCACCT TAtcTAAAAG GTGGTAAAG291 E. coliTa cgaTGctctT gAggtgcaaa ATggtaatgA gcgtcTgGTg...TAAaGcgA CTcTGGGcCG TaTCaTgAAC GTaC...CcT gaTgtgtCCg TtCgctAAgG GcGGTAAAG2771 L. monocytogenesTa tAaatctgAT gcAgAaGaaG caccaaCtag ccAAcTtact...TAcaGtaA CTcTtGGTCG TGTaTTtAAt GTat...CTT gcTAGCtCCT TACTTAAAAG GTGGTAAAa324 S. aureusgT tATTGatgtg cctAAaGaaG AaggtaCAat AcAAcTAacA...TgAtGAaA CaTTaGGTCG TGTaTTtAAt GTaC...tTT AcTAGCACCT TAtaTtAAAG GTGGTAAAa366 S. epidermidisca cATcGaagtT cctAAaGaaG ATggagCgCt tcAAtTAacA...TgAcGtaA CTcTaGGaaG aGTgTTtAAC GTaC...CTT ATTAGCACCT TACaTAAAAG GTGGTAAAa370 Selected        ATTGTCTAT AAAAATGGCG ATAAGTC 627 sequences                  AAAATGGCG ATAAGTCACA AAAAGTA 628 for species- specificprimer Selected                                                    GGATA CTTTGGGTCG TGTCTTCAAC G625 sequences                                                                                           ATTAGCACCT TACTTAAAAG GTGGTA626 for species- specific primers^(g) The sequence numbering refers tothe Streptococcus agalactiae tuf gene fragment (SEQ ID NO. 380).Nucleotides in capitals are identical to the selected sequences or matchthose sequences. Mismatches are indicated by lower-case letters. Dotsindicate gaps in the sequences displayed. ^(a,d,e,f)These sequences wereobtained from Genbank and have accession #: a = AB009314, d = AF001955,e = U31170, and f = V00311. ^(b,c)These sequences were obtained fromgenome sequencing projects. ^(g)These sequences are thereverse-complement of the selected primers.

TABLE 49Strategy for the selection of Candida albicans/dubliniensis-specificamplification primers, Candida albicans-specific hybridization probe andCandida dubliniensis-specific hybridization probe from tuf sequences.SEQ ID Accession337                             368   403                      428   460                              491NO.: #: C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T624 — C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T409 — C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T410 — C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T407 — C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T408 — C. dubliniensisCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC CACCAACT...C AAATCCGGTA AgGTTACTGG TAAGACCTTG T412 — C. dubliniensisCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC CACCAACT...C AAATCCGGTA AgGTTACTGG TAAGACCTTG T414 — C. dubliniensisCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC CACCAACT...C AAATCCGGTA AgGTTACTGG TAAGACCTTG T415 — C. glabrataCATC AAGAAGGTcG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCcaC CACCAACG...C AAggCtGGTg tcGTcAagGG TAAGACCTTG T417 — C. guilliermondiiCGTC AAGAAGGTTG GTTACAACCC tAAGACTG...CAACATGA TTGAggCtTC tACCAACT...C AAggCtGGTA AgtccACcGG TAAGACtTTG T418 — C. kefyrCATC AAGAAGGTcG GTTACAACCC AAAGAATG...CAACATGA TTGAAgCcaC CACCAACG...C AAggCtGGTA ccGTcAagGG TAAGACCTTG T421 — C. kruseiCATC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCATC CACCAACT...C AAggCaGGTg ttGTTAagGG TAAGACCTTA T422 — C. lusitaniaeCGTC AAGAAGGTTG GTTACAACCC tAAGACTG...CAACATGA TTGAgCCATC YACCAACT...C AAgTCYGGTA AgtccACcGG TAAGACCTTG T424 — C. neoformansCATC AAGAAGGTTG GTTACAACCC cAAGgCTG...CAACATGt TgGAggagaC CACCAAGT...C AAgTCtGGTg tttccAagGG TAAGACCcTC C623 — C. parapsilosisCGTC AAGAAGGTTG GTTACAACCC tAAagCTG...CAAtATGA TTGAACCATC aACCAACT...T AAAgCtGGTA AgGTTACcGG TAAGACCTTG T426 — C. tropicalisCGTC AAGAAGGTTG GTTACAACCC tAAGgCTG...CAACATGA TTGAAgCtTC tACCAACT...C AAggCtGGTA AgGTTACcGG TAAGACtTTG T429 — A. fumigatusCATC AAGAAGGTcG GcTACAACCC cAAGgCCG...CAACATGc TTGAgCCcTC CtCCAACT...C AAggCCGGcA AgGTcACTGG TAAGACCcTC A404 — HumanCATt AAGAAaaTTG GcTACAACCC cgAcACAG...CAACATGc TgGAgCCAag tgCtAACA...T AAggatGGcA AtGccAgTGG aAccACgcTG C2741 X03558 P. anomalaTATC AAGAAaGTTG GTTACAACCC AAAaACTG...TAACATGA TTGAACCATC aWCtAACT...C AAAgCtGGTg AAGcTAaaGG TAAaACtTTA T447 — S. cerevisiaeTATC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtaC CACCAACG...C AAggCCGGTg tcGTcAagGG TAAGACtTTG T622 — S. pombeCATC AAGAAGGTcG GTTtCAACCC cAAGACCG...TAACATGA TTGAgCCcaC CACCAACA...C AAggCtGGTg tcGTcAagGG TAAGACtcTT T2759 U42189 Selected sequence    C AAGAAGGTTG GTTACAACCC AAAGAfor species-specific amplification primer^(a) Selected sequence                                                                        ATCCGGTA AAGTTACTGG TAAGACCTfor species-specific amplification primer^(a,b) Selected sequences                                         CATGA TTGAACCATC CACCA (C. albicans)577 for species-specific                                         CATGA TTGAAGCTTC CACCA (C. dubliniensis)578 hybridization probes The sequence numbering refers to the Candidaalbicans tuf gene fragment (SEQ ID NO. 408). Nucleotides in capitals areidentical to the selected sequences or match those sequences. Mismatchesfor SEQ ID NO. 577 are indicated by lower-case letters. Mismatches forSEQ ID NO. 578 are indicated by underlined nucleotides. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a) C. albicans primers have been described in a previouspatent (publication WO98/20157, SEQ ID NOs. 11-12) ^(b)This sequence isthe reverse-complement of the selected primer.

TABLE 50Strategy for the selection of Staphylococcus-specific amplification primers from tuf sequences.310                            340   652                            682SEQ ID NO.: Accession #: S. aureusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTACTG AAATGGTAAT GC179 — S. aureusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACC~ ~~~~~~~~~~ ~~~~~~~~~~ GC176 — S. aureusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTMCTG AAATGGTAAT GC177 — S. aureus aureusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTACTG AAATGGTAAT GC180 — S. auricularisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...ActTTACCA GAAGGTACaG AAATGGTAAT GC181 — S. capitis capitisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC182 — M. caseolyticusA CTGGaCGTGT TGAgCGTGGa CAAgTtAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC183 — S. cohniiA CAGGgCGTGT TGAACGTGGT CAAATCAAAG...ActTTACCA GAAGGTACTG AAATGGTTAT GC184 — S. epidermidisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~185 — S. epidermidisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACaG AAATGGTTAT GC2859^(a) — S. haemolyticusA CAGGCCGTGT TGAACGTGGg CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC186 — S. haemolyticusA CAGGtCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAG~~~~~~ ~~~~~~~~~~ ~~188 — S. haemolyticusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGG~~~~ ~~189 — S. hominis hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC191 — S. hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC193 — S. hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGG~~~~~ ~~~~~~~~~~ ~~194 — S. hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC195 — S. hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC196 — S. lugdunensisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACaG AAATGGTTAT GC197 — S. saprophyticusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~198 — S. saprophyticusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC199 — S. saprophyticusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC200 — S. sciuri sciuriA CAGGCCGTGT TGAACGTGGT CAAATCACTG...AACTTACCA GAAGGTACTG AAATGGTTAT GC201 — S. warneriA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CAaTTACCA GAAGGTACTG ~~~~~~~~~~ ~~187 — S. warneriA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~192 — S. warneriA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CAaTTACCA GAAGGTACTG AAATGGTTAT GC202 — B. subtilisA CTGGCCGTGT aGAACGcGGa CAAgTtAAAG...CAtcTtCCA GAAGGcgtaG AAATGGTTAT GC2763 Z99104 E. coliA CCGGtCGTGT aGAACGcGGT atcATCAAAG...GAacTgCCg GAAGGcgtaG AgATGGTAAT GC78 — L. monocytogenesA CTGGaCGTGT TGAACGTGGa CAAgTtAAAG...AcacTtCCA GAAGGTACTG AAATGGTAAY GC2857^(c) — Selected sequence for genus-specific primer    GGCCGTGT TGAACGTGGT CAAATCA 553Selected sequences for genus-specific primers^(b)                                        TTACCA GAAGGTACTG AAATGGTIA 575                                        TTACCA GAAGGTACTG AAATGGTWA 707The sequence numbering refers to the Staphylococcus aureus tuf genefragment (SEQ ID NO. 179). Nucleotides in capitals are identical to theselected sequences or match those sequences. Mismatches are indicated bylower-case letters. “~” indicate incomplete sequence data. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a)This sequence corresponds to SEQ ID No. 141 of patentpublication WO98/20157. ^(b)These sequences are the reverse-complementof the selected primers. ^(c)This sequence corresponds to SEQ ID NO. 138of patent publication WO98/20157.

TABLE 51 Strategy for the selection of theStaphylococcus-specific hybridization probe from tuf sequences.400                       425 SEQ ID NO.: Accession #: S. aureusG TTGAAATGTT CCGTAAATTA TTAGA 179 — S. aureusG TTGAAATGTT CCGTAAATTA TTAGA 176 — S. aureusG TTGAAATGTT CCGTAAATTA TTAGA 177 — S. aureusG TTGAAATGTT CCGTAAATTA TTAGA 178 — S. aureus aureusG TTGAAATGTT CCGTAAATTA TTAGA 180 — S. auricularisG TAGAAATGTT CCGTAAATTA TTAGA 181 — S. capitis capitisG TAGAAATGTT CCGTAAATTA TTAGA 182 — M. caseolyticusG TAGAAATGTT CCGTAAATTA TTAGA 183 — S. cohniiG TAGAAATGTT CCGTAAATTA TTAGA 184 — S. epidermidisG TAGAAATGTT CCGTAAATTA TTAGA 185 — S. haemolyticusG TAGAAATGTT CCGTAAATTA TTAGA 186 — S. haemolyticusG TAGAAATGTT CCGTAAATTA TTAGA 189 — S. haemolyticusG TAGAAATGTT CCGTAAATTA TTAGA 190 — S. haemolyticusG TAGAAATGTT CCGTAAATTA TTAGA 188 — S. hominisG TAGAAATGTT CCGTAAATTA TTAGA 196 — S. hominisG TAGAAATGTT CCGTAAATTA TTAGA 194 — S. hominis hominisG TAGAAATGTT CCGTAAATTA TTAGA 191 — S. hominisG TAGAAATGTT CCGTAAATTA TTAGA 193 — S. hominisG TAGAAATGTT CCGTAAATTA TTAGA 195 — S. lugdunensisG TAGAAATGTT CCGTAAATTA TTAGA 197 — S. saprophyticusG TAGAAATGTT CCGTAAATTA TTAGA 198 — S. saprophyticusG TAGAAATGTT CCGTAAATTA TTAGA 200 — S. saprophyticusG TAGAAATGTT CCGTAAATTA TTAGA 199 — S. sciuri sciuriG TTGAAATGTT CCGTAAATTA TTAGA 201 — S. warneriG TAGAAATGTT CCGTAAgTTA TTAGA 187 — S. warneriG TAGAAATGTT CCGTAAgTTA TTAGA 192 — S. warneriG TAGAAATGTT CCGTAAgTTA TTAGA 202 — S. warneriG TAGAAATGTT CCGTAAgTTA TTAGA 203 — B. subtilisG TTGAAATGTT CCGTAAgcTt cTTGA 2763 Z99104 E. coliG TTGAAATGTT CCGcAAAcTg cTGGA 78 — L. monocytogenesG TAGAAATGTT CCGTAAATTA cTAGA 2857^(a) — Selected sequence for    GAAATGTT CCGTAAATTA TT 605 genus-specific hybridization probe Thesequence numbering refers to the Staphylococcus aureus tuf gene fragment(SEQ ID NO. 179). Nucleotides in capitals are identical to the selectedsequence or match that sequence. Mismatches are indicated by lower-caseletters. ^(a)This sequence corresponds to SEQ ID NO. 138 of patentpublication WO98/20157.

TABLE 52Strategy for the selection of Staphylococcus saprophyticus-specific and ofStaphylococcus haemolyticus-specific hybridization probes from tuf sequences.SEQ ID 339                                            383 NO.: S. aureusAG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA 179 S. aureusAG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA 176 S. aureusAG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA 177 S. aureusAG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA 178 S. aureus aureusAG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA 180 S. auricularisAG TCGGTGAAGA AgTtGAAATC ATcGGTATga AaGACggTTC AAA 181S. capitis capitis AG TtGGTGAAGA AgTtGAAATC ATcGGTATCC AcGAaACTTC TAA182 M. caseolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTtTaa cTGAagaacC AAA183 S. cohnii AG TCGGTGAAGA AgTtGAAATC ATcGGTATgC AaGAagaTTC CAA 184S. epidermidis AG TtGGTGAAGA AgTtGAAATC ATcGGTATgC AcGAaACTTC TAA 185S. haemolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA 186S. haemolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA 189S. haemolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA 190S. haemolyticus AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA 188S. hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA 194S. hominis hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA191 S. hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA 193S. hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA 195S. hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAtACTTC TAA 196S. lugdunensis AG TCGGTGAAGA AgTtGAAATt ATTGGTATCC AcGAtACTaC TAA 197S. saprophyticus AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA 198S. saprophyticus AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA 200S. saprophyticus AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA 199S. sciuri sciuri TG TtGGTGAAGA AgTtGAAATC ATcGGTtTaa cTGAagaaTC TAA 201S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA 187S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA 192S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA 202S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA 203B. subtilis AG TCGGTGAcGA AgTtGAAATC ATcGGTcTtC AaGAagagag AAA 2772E. coli AG TtGGTGAAGA AgTtGAAATC gTTGGTATCa AaGAgACTca GAA 78L. monocytogenes AG TtGGTGAcGA AgTaGAAgTt ATcGGTATCg AaGAagaaag AAA2857^(b) Selected sequences for    CGGTGAAGA AATCGAAATC A (S. saprophyticus) 599 species-specific       (S. haemolyticus) ATTGGTATCC ATGACACTTC 594 hybridization probesThe sequence numbering refers to the Staphylococcus aureus tuf genefragment (SEQ ID NO. 179). Nucleotides in capitals are identical to theselected sequences or match those sequences. Mismatches are indicated bylower-case letters. ^(a)This sequence was obtained from Genbankaccession #Z99104. ^(b)This sequence corresponds to SEQ ID NO. 138 ofpatent publication WO98/20157.

TABLE 53Strategy for the selection of Staphylococcus aureus-specific and ofStaphylococcus epidermidis-specific hybridization probes from tuf sequences.SEQ ID 521                      547   592                      617 NO.:S. aureus TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT179 S. aureusTACACCACA TACTGAATTC AAAGCAG...TTCTTCtC~ ~~~~~~~~~~ ~~~~~~~ 178S. aureus TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT176 S. aureusTACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT 177S. aureus aureusTACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT 180S. auricularisTACACCACA cACTaAATTC ActGCAG...TTCTTCtCT AACTAcCGtC CACAATT 181S. capitis capitisCACACCACA cACTaAATTC AAAGCGG...TTCTTCAgT AACTAcCGCC CACAATT 182M. caseolyticusTACtCCACA TACTaAATTC AAAGCTG...TTCTTCACT AACTAcCGCC CtCAGTT 183S. cohnii TACACCACA cACaaAcTTt AAAGCGG...TTCTTCAgT AACTATCGCC CACAATT184 S. epidermidisTACACCACA cACaaAATTC AAAGCTG...TTCTTCACT AACTATCGCC CACAATT 185S. haemolyticusCACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtC CACAATT 186S. haemolyticusCACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtC CACAATT 189S. haemolyticusCACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtC CACAATT 190S. haemolyticusTACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtC CACAATT 188S. hominis CACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtC CACAATT195 S. hominisTACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtC CACAATT 196S. hominis hominisTACACCtCA cACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtC CACAATT 191S. hominis TACACCtCA cACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtC CACAATT193 S. hominisTACACCtCA cACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtC CACAATT 194S. lugdunensisTACACCtCA cACTaAATTt AAAGCTG...TTCTTCtCa AACTAcCGCC CACAATT 197S. saprophyticusTACACCACA TACaaAATTC AAAGCGG...TTCTTCACT AACTAcCGCC CACAATT 198S. saprophyticusTACACCACA TACaaAATTC AAAGCGG...TTCTTCACT AACTAcCGCC CACAATT 199S. saprophyticusTACACCACA TACaaAATTC AAAGCGG...TTCTTCACT AACTAcCGCC CACAATT 200S. sciuri sciuriCACACCtCA cACTaAATTC AAAGCTG...TTCTTCACa AACTAcCGCC CACAATT 201S. warneri TACACCACA TACaaAATTC AAAGCGG...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~192 S. warneriTACACCACA TACaaAATTC AAAGCGG...TTCTTCAgT AACTAcCGCC CACAATT 187S. warneri TACACCACA TACaaAATTC AAAGCGG...TTCTTCAgT AACTAcCGCC CACAATT202 S. warneriTACACCACA TACaaAATTC AAAGCGG...TTCTTCAgT AACTAcCGCC CACAATT 203B. subtilis CACtCCACA cAgcaAATTC AAAGCTG...TTCTTCtCT AACTAcCGtC CtCAGTT2772 E. coli CAAgCCgCA cACcaAgTTC gAAtCTG...TTCTTCAaa ggCTAcCGtC CgCAGTT78 L. monocytogenesTACtCCACA cACTaAcTTC AAAGCTG...TTCTTCAac AACTAcCGCC CACAATT 2857^(b)Selected sequences    ACCACA TACTGAATTC AAAG (S. aureus) 585for species-specific                 (S. epidermidis) TTCACT AACTATCGCC CACA 593hybridization probes The sequence numbering refers to the Staphylococcusaureus tuf gene fragment (SEQ ID NO. 179). Nucleotides in capitals areidentical to the selected sequences or match those sequences. Mismatchesare indicated by lower-case letters. “~” indicate incomplete sequencedata. Dots indicate gaps in the sequences displayed. ^(a)This sequencewas obtained from Genbank accession #Z99104. ^(b)This sequencecorresponds to SEQ ID NO. 138 of previous patent publication WO98/20157.

TABLE 54 Strategy for the selection of the Staphylococcus hominis-specific hybridization probe from tuf sequences.358                       383 SEQ ID NO.: S. aureusATC ATcGGTtTac AtGAcACaTC TAA 179 S. aureusATC ATcGGTtTac AtGAcACaTC TAA 176 S. aureusATC ATcGGTtTac AtGAcACaTC TAA 177 S. aureusATC ATcGGTtTac AtGAcACaTC TAA 178 S. aureus aureusATC ATcGGTtTac AtGAcACaTC TAA 180 S. auricularisATC ATcGGTATgA AAGAcggTTC AAA 181 S. capitis capitisATC ATcGGTATCc AcGAAACTTC TAA 182 M. caseolyticusATC ATTGGTtTaA ctGAAgaacC AAA 183 S. cohniiATC ATcGGTATgc AAGAAgaTTC CAA 184 S. epidermidisATC ATcGGTATgc AcGAAACTTC TAA 185 S. haemolyticusATC ATTGGTATCc AtGAcACTTC TAA 186 S. haemolyticusATC ATTGGTATCc AtGAcACTTC TAA 189 S. haemolyticusATC ATTGGTATCc AtGAcACTTC TAA 190 S. haemolyticusATT ATTGGTATCA AAGAAACTTC TAA 188 S. hominisATT ATTGGTATCA AAGAtACTTC TAA 196 S. hominisATT ATTGGTATCA AAGAAACTTC TAA 194 S. hominis hominisATT ATTGGTATCA AAGAAACTTC TAA 191 S. hominisATT ATTGGTATCA AAGAAACTTC TAA 193 S. hominisATT ATTGGTATCA AAGAAACTTC TAA 195 S. lugdunensisATT ATTGGTATCc AcGAtACTaC TAA 197 S. saprophyticusATC ATcGGTATgc AAGAAgaaTC CAA 198 S. saprophyticusATC ATcGGTATgc AAGAAgaaTC CAA 200 S. saprophyticusATC ATcGGTATgc AAGAAgaaTC CAA 199 S. sciuri sciuriATC ATcGGTtTaA ctGAAgaaTC TAA 201 S. warneriATC ATcGGTtTac AtGAcACTTC TAA 187 S. warneriATC ATcGGTtTac AtGAcACTTC TAA 192 S. warneriATC ATcGGTtTac AtGAcACTTC TAA 202 S. warneriATC ATcGGTtTac AtGAcACTTC TAA 203 B. subtilisATC ATcGGTcTtc AAGAAgagag AAA 2772 E. coli ATC gTTGGTATCA AAGAgACTca GAA78 L. monocytogenes GTT ATcGGTATCg AAGAAgaaag AAA 2857^(b)Selected sequence for     ATTGGTATCA AAGAAACTTC 597 species-specifichybridization probe The sequence numbering refers to the Staphylococcusaureus tuf gene fragment (SEQ ID NO. 179). Nucleotides in capitals areidentical to the selected sequences or match those sequences. Mismatchesare indicated by lower-case letters. Dots indicate gaps in the sequencesdisplayed. ^(a)This sequence was obtained from Genbank accession#Z99104. ^(b)This sequence corresponds to SEQ ID NO. 138 of previouspatent publication WO98/20157.

TABLE 55Strategy for the selection of the Enterococcus-specific amplificationprimers from tuf sequences.270                         298   556                        582SEQ ID NO.:  Accession #:  E. aviumTAGAATTAAT GGCTGCTGTT GACGAATAT...TGAA GATATCCAAC GTGGACAAGT ATT2855^(a) — E. casseliflavusTGGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT 58 —E. cecorumTAGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GATATCCAAC GTGGtCAAGT ATT 59 —E. disparTAGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GATATCCAAC GTGGtCAAGT ATT 60 —E. duransTTGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT TTT 61 —E. flavescensTGGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT 65 —E. faeciumTTGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT TTT 608 —E. faecalisTAGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GATATCGAAC GTGGACAAGT ATT 607 —E. gallinarumTGGAATTgAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT 609 —E. hiraeTTGAATTgAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT TTT 67 —E. mundtiiTTGAATTgAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGtCAAGT TTT 68 —E. pseudoaviumTAGAATTAAT GSCTGCTGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT 69 —E. raffinosusTAGAATTAAT GGCTGCTGTT GATGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT 70 —E. saccharolyticusTCGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT ATT 71 —E. solitariusTGGAcTTAAT GGaTGCAGTT GATGAcTAC...TGAt GATATCGAAC GTGGtCAAGT ATT 72 —E. coli TGGAAcTggc tGgcttccTg GATtctTAY...TGAA GAaATCGAAC GTGGtCAgGT ACT78 — B. cepaciaTGAgccTggc cGacGCgcTg GACacgTAC...TGAA GACgTgGAgC GTGGcCAgGT TCT 16 —B. fragilisTGGAAcTgAT GGaaGCTGTT GATactTGG...GAAc GAaATCaAAC GTGGtatgGT TCT 2736M22247 B. subtilisTCGAAcTtAT GGaTGCgGTT GATGAgTAC...TGAA GAaATCCAAC GTGGtCAAGT ACT 2763Z99104 C. diphtheriaeTCGAccTcAT GcagGCTtgc KATGAtTCC...CGAA GACgTtGAgC GTGGcCAgGT TGT 662 —C. trachomatisGAGAgcTAAT GcaaGCcGTc GATGAtAAT...GAAc GATgTgGAAa GaGGAatgGT TGT 22 —G. vaginalisAGGAAcTcAT GaagGCTGTT GACGAgTAC...TACc GACgTtGAgC GTGGtCAgGT TGT2856^(c) — S. aureusTAGAATTART GGaaGCTGTa GATactTAC...TGAA GACgTaCAAC GTGGtCAAGT ATT 179 —S. pneumoniaeTGGAATTgAT GaacaCAGTT GATGAgTAT...TGAt GAaATCGAAC GTGGACAAGT TAT2861^(d) — A. adiacensTAGAATTAAT GGCTGCTGTT GACGAATAC...TGAA aACATCGAAC GTGGACAAGT TCT2854^(e) — G. haemolysansTCGAATTAAT GGaaaCAGTT GACGAATAC...TGAA GACATCGAAC GTGGACAAGT TTT 87 —G. morbillorumTCGAATTAAT GGaaaCAGTT GACGAgTAC...TGAA GATATCGAAC GTGGACAAGT TTT 88 —Selected sequence for amplification primer    AATTAAT GGCTGCWGTT GAYGAA1137   Selected sequence for amplification primer^(b)                                     A GAYATCSAAC GTGGACAAGT 1136   Thesequence numbering refers to the Enterococcus durans tuf gene fragment(SEQ ID NO. 61). Nucleotides in capitals are identical to the selectedsequences or match those sequences. Mismatches are indicated bylower-case letters. Dots indicate gaps in the sequences displayed. “Y”“W” and “S” designate nucleotide positions which are degenerated. “Y”stands for C or T; “W” stands for A or T; “S” stands for C or G. “I”stands for inosine which is a nucleotide analog that can bind to any ofthe four A, C, G or T. ^(a)This sequence corresponds to SEQ ID NO. 131of previous patent publication WO98/20157. ^(b)This sequence is thereverse-complement of the selected primer. ^(c)This sequence correspondsto SEQ ID NO. 135 of previous patent publication WO98/20157. ^(d)Thissequence corresponds to SEQ ID NO. 145 of previous patent publicationWO98/20157. ^(e)This sequence corresponds to SEQ ID NO. 118 of previouspatent publication WO98/20157.

TABLE 56Strategy for the selection of the Enterococcus faecalis-specifichybridization probe, of the Enterococcus faecium-specific hybridizationprobe and of the Enterococcus casseliflavus-flavescens-gallinarum group-specific hybridization probe from tuf sequences.395                                                     448...526                    549SEQ ID NO.: Accession #: E. aviumGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATcGCT...CATc GGTGCtTTGt TACGTGGTGT2855^(a) — E. casseliflavusGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT GGTATTGCT...CATT GGTGCATTGC TACGTGGTGT58 — E. cecorumGTTGA ACGTGGacAA GTaCGtGTTG GTGACGAAGT TGAAaTaGTT GGTATcCAT...CATc GGTGCATTat TACGTGGTGT59 — E. disparGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATcGCT...CATT GGTGCATTat TACGTGGTGT60 — E. duransGTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAtaTcGTT GGTATcGCA...CATT GGTGCtTTaC TACGTGGTGT61 — E. faecalisGTTGA ACGTGGTGAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT GGTATTAAA...CTTc GGTGCtTTat TACGTGGTGT62 — E. faeciumGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAGTTGTT GGTATTGCT...CATT GGTGCtTTaC TACGTGGTGT608 — E. flavescensGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT GGTATTGCT...CATT GGTGCATTGC TACGTGGGGT65 — E. gallinarumGTTGA ACGTGGacAA GTTCGCGTTG GTGATGAAGT aGAAaTcGTT GGTATTGCT...CATT GGTGCATTGC TACGTGGGGT609 — E. hiraeGTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAtaTcGTT GGTATcGCA...CATT GGTGCtTTaC TACGTGGTGT67 — E. mundtiiGTTGA ACGTGGacAA GYTCGtGTTG GTGACGttaT cGAtaTcGTT GGTATcGCA...CATT GGTGCgTTaC TACGTGGTGT68 — E. pseudoaviumGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATCGCT...CATc GGTGCATTat TACGTGGTGT69 — E. raffinosusGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATTGCT...CATT GGTGCATTat TACGTGGTGT70 — E. saccharolyticusGTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAAaTcGTT GGTATcGAC...CATc GGTGCtTTat TACGTGGGGT71 — E. solitariusGTTGA ACGcGGgact aTcaaaGTCG GCGATGAAGT TGAcaTTaTT GGTATTCAT...CATT GGTaCtTTGt TACGTGGTGT72 — C. diphtheriaeGTTGA gCGTGGctcc cTgaagGTCA ACGAGGAcGT cGAgaTcaTc GGTATcCGC...CTGT GGTctgcTtC TcCGTGGCGT662 — G. vaginalisGTTGA gCGTGGTaAg cTcCcaATCA ACACCCcAGT TGAgaTcGTT GGTtTgCGC...CACT GGTcttcTtC TcCGcGGTAT2856^(b) — B. cepaciaGTCGA gCGcGGcatc GTgaagGTCG GCGAAGAAaT cGAAaTcGTc GGTATcAAG...CGTT GGTatccTGC TgCGcGGCAC16 — S. aureusGTTGA ACGTGGTcAA aTcaaaGTTG GTGAAGAAGT TGAAaTcaTc GGTtTaCAT...CATT GGTGCATTat TACGTGGTGT179 — B. subtilisGTAGA ACGcGGacAA GTTaaaGTCG GTGACGAAGT TGAAaTcaTc GGTcTTCAA...CATT GGTGCccTtC TtCGcGGTGT2763 Z99104 S. pneumoniaeATCGA cCGTGGTatc GTTaaaGTCA ACGACGAAaT cGAAaTcGTT GGTATcAAA...CGTa GGTGtccTtC TtCGTGGTGT2861^(c) — E. coliGTAGA ACGcGGTatc aTcaaaGTTG GTGAAGAAGT TGAAaTcGTT GGTATcAAA...CGTa GGTGttcTGC TgCGTGGTAT78 — B. fragilisATCGA AacTGGTGtt aTcCatGTAG GTGATGAAaT cGAAaTccTc GGTtTgGGT...CGTa GGTctgTTGC TtCGTGGTGT2736 M22247 C. trachomatisATTGA gCGTGGaatt GTTaaaGTTT CCGATAAAGT TcAgtTgGTc GGTcTTAGA...CGTT GGattgcTcC TcaGaGGTAT22 — Selected sequences for    GA ACGTGGTGAA GTTCGC (E. faecalis) 1174species-specific or                                  AAGT TGAAGTTGTT GGTATT (E. faecium)602 group-specific                                                                  T GGTGCATTGC TACGTGG1122 hybridization probes The sequence numbering refers to theEnterococcus faecium tuf gene fragments (SEQ ID NO. 608). Nucleotides incapitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)This sequence corresponds to SEQ IDNO. 131 of previous patent publication WO98/20157. ^(b)This sequencecorresponds to SEQ ID NO. 135 of previous patent publication WO98/20157.^(c)This sequence corresponds to SEQ ID NO. 145 of previous patentpublication WO98/20157.

TABLE 57 Strategy for the selection of primers for the identification ofplatelets contaminants from tuf sequences. SEQ ID Accession467                          495   689                          717 NO.:#: B. cereusGTA ACTGGTGTaG AGATGTTCCG TAAACT...C AGTTCTACTT CCGTACAACT GACGTAAC   7— B. subtilisGTT ACaGGTGTTG AAATGTTCCG TAAGCT...C AGTTCTACTT CCGTACAACT GACGTAAC 2763Z99104 E. cloacaeTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GACGTGAC  54— E. coliTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACTACT GACGTGAC  78— K. oxytocaTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GACGTGAC 100— K. pneumoniaeTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACTACT GACGTGAC 103— P. aeruginosaTGC ACcGGCGTTG AAATGTTCCG CAAGCT...C AGTTCTACTT CCGTACCACK GACGTGAC 153— S. agalactiaeGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT CCGTACAACT GACGTAAC 209— S. aureusGTT ACaGGTGTTG AAATGTTCCG TAAATT...C AATTCTATTT CCGTACTACT GACGTAAC 2858^(a) — S. choleraesuisTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACTACT GACGTGAC 159— S. epidermidisGTT ACTGGTGTaG AAATGTTCCG TAAATT...C AATTCTATTT CCGTACTACT GACGTAAC 611— S. marcescensTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACCACT GACGTGAC 168— S. mutansGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT CCGTACAACT GACGTAAC 224— S. pyogenesGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT CCGTACAACT GACGTAAC 993U40453 S. salivariusGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AGTTCTACTT CCGTACAACT GACGTAAC 2862^(c) — S. sanguinisGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AGTTCTACTT CCGTACAACT GACGTTAC 227— Y. TGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GAtGTAAC235 — enterocolitica Selected     ACTGGYGTTG AIATGTTCCG YAA 636sequence for amplification primer Selected                                       TTCTAYTT CCGTACIACT GACGT 637sequence for amplification primer^(b) The sequence numbering refers tothe E. coli tuf gene fragment (SEQ ID NO. 78). Nucleotides in capitalsare identical to the selected sequences or match those sequences.Mismatches are indicated by lower-case letters. Dots indicate gaps inthe sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a)This sequence corresponds to SEQ ID NO. 140 of previouspatent publication WO98/20157. ^(b)This sequence is thereverse-complement of the selected primer. ^(c)This sequence correspondsto SEQ ID NO. 146 of previous patent publication WO98/20157.

TABLE 58Strategy for the selection of the universal amplification primers from atpD sequences.616                                        657   781                             812SEQ ID NO. ACCESSION #: C. glutamicum GTGT T CGGTC AGATG GATGA GCCACCAGGA  GT CCGTATG CGC...CGTATg CCTTCCGCCG TGGGTTACCA GCCAAC2773 X76875 M. tuberculosis GTAT T CGGAC AGATG G ACGA GCCGCCGGGC a CCCGTATG CGT...CGGATg CCGTCGGCCG TGGGATACCA GCCCAC 2735 Z73419E. faecalis GTGT T CGGAC AAATGAACGA ACCACCAGGT  GCTCGGATG CGG...CGTATg CCTTCTGCCG TTGGTTACCA ACCAAC 291 — S. agalactiaeGTCT T TGGTC AAATGAATGA ACCACCAGGA  GCACGTATG CGT...CGTATg CCTTCAGCCG TTGGTTATCA ACCAAC 380 — B. subtilis GTATT CGGAC AAATGAACGA GCCGCCGGGC  GCACGTATG CGT...CGTATg CCTTCAGCGG TTGGTTATCA GCCGAC 2774 Z28592L. monocytogenes GTAT T CGGTC AAATGAACGA GCCACCAGGT  GCGCGTATG CGT...CGTATg CCATCTGCGG TAGGTTACCA ACCAAC 324 — S. aureus GTAT TCGGGC AAATGAATGA GCCACCTGGT  GCACGTATG CGT...CGTATg CCTTCTGCAG TAGGTTACCA ACCAAC 366 — A. baumanniiGTCTACGGTC AGATGAACGA GCCACCAGGT aaCCGTtTa CGC...CGTATg CCATCTGCGG TAGGTTACCA ACCTAC243 — N. gonorrhoeaeGTGTATGGCC AAATGAACGA ACCTCCAGGC aaCCGTcTG CGC...CGTATg CCTTCTGCAG TGGGTTACCA ACCGAC2775 Genome project C. freundiiGTATATGGCC AGATGAACGA GCCGCCTGGA aaCCGTcTG CGT...CGTATg CCATCAGCGG TAGGCTACCA GCCGAC264 — E. cloacaeGTTTACGGCC AGATGAACGA GCCACCAGGA aaCCGTcTG CGC...CGTATg CCTTCAGCGG TAGGTTATCA GCCTAC284 — E. coliGTGTATGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg CCTTCAGCGG TAGGTTATCA GCCGAC669 V00267 S. typhimuriumGTGTATGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg CCTTCCGCAG TAGGTTACCA GCCGAC351 — K. pneumoniaeGTGTACGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg CCTTCAGCGG TAGGTTATCA GCCGAC317 — S. marcescensGTTTACGGCC AGATGAACGA GCCACCAGGT aaCCGTcTG CGC...CGTATg CCATCCGCGG TAGGTTATCA GCCAAC357 — Y. enterocoliticaGTTTATGGCC AAATGAATGA GCCACCAGGT aaCCGTcTG CGC...CGTATg CCATCTGCCG TAGGTTACCA GCCAAC393 — B. cepaciaGTGTACGGCC AGATGAACGA GCCGCCGGGC aaCCGTcTG CGC...CGTATg CCGTCGGCAG TGGGCTATCA GCCGAC2728 X76877 H. influenzaeGTTTATGGTC AAATGAACGA GCCACCAGGT aaCCGTtTa CGT...CGTATg CCATCCGCGG TAGGTTACCA ACCGAC2776 U32730 M. pneumoniae GTGT T TGGTC AGATGAACGA ACCCCCAGGA  GCACGGATG CGG...CGGATg CCATCAGCCG TGGGTTACCA ACCAAC 2738 U43738 H. pyloriTGCTATGGGC AAATGAATGA GCCACCAGGT  GCAAGGAat CGC...CGTATC CCTTCAGCGG TGGGGTATCA GCCCAC 670 V00267 B. fragilisGTGT T CGGAC AGATGAACGA ACCTCCTGGA  GCACGTgct TCA...CGTATg CCTTCTGCGG TAGGTTATCA ACCTAC 2736 M22247Selected sequences for            C ARATGRAYGA RCCICCIGGI GYIMGIATG 562universal primers    TAYGGIC ARATGAAYGA RCCICCIGGI AA 564Selected sequences for                                                     ATH CCITCIGCIG TIGGITAYCA RCC565 universal primers^(a)                                                    ATG CCITCIGCIG TIGGITAYCA RCC563 The sequence numbering refers to the Escherichia coli atpD genefragment (SEQ ID NO. 669). Nucleotides in capitals are identical to theselected sequences or match those sequences. Mismatches for SEQ ID NOs.562 and 565 are indicated by lower-case letters. Mismatches for SEQ IDNOs. 564 and 563 are indicated by underlined nucleotides. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” lettersdesignate nucleotide positions which are degenerated. “R” stands for Aor G; “Y” stands for C or T; “M” stands for A or C; “K” stands for G orT; “W” stands for A or T; “H” stands for A, C or T; “S” stands for C orG. “I” stands for inosine which is a nucleotide analog that can bind toany of the four nucleotides A, C, G or T. ^(a)These sequences are thereverse-complement of the selected primers.

TABLE 59 Specific and ubiquitous primers for nucleic acidamplification (recA sequences). Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionUniversal primers (recA) 919 5′-GGI CCI GAR TCI TMI GGI AAR AC 918^(a)437-459  920^(b) 5′-TCI CCV ATI TCI CCI TCI AIY TC 918^(a) 701-723 9215′-TIY RTI GAY GCI GAR CAI GC 918^(a) 515-534  922^(b)5′-TAR AAY TTI ARI GCI YKI CCI CC 918^(a) 872-894Sequencing primers (recA) 1605  5′-ATY ATY GAA RTI TAY GCI CC 1704^(a) 220-239 1606  5′-CCR AAC ATI AYI CCI ACT TTT TC 1704^(a)  628-650Universal primers (rad51) 935 5′-GGI AAR WSI CAR YTI TGY CAY AC 939^(a)568-590  936^(b) 5′-TCI SIY TCI GGI ARR CAI GG 939^(a) 1126-1145Universal primers (dmc1) 937 5′-ATI ACI GAR GYI TTY GGI GAR TT 940^(a)1038-1060  938^(b) 5′-CYI GTI GYI SWI GCR TGI GC 940^(a) 1554-1573^(a)Sequences from databases. ^(b)These sequences are from thecomplementary DNA strand of the sequence of the originating fragmentgiven in the Sequence Listing.

TABLE 60 Specific and ubiquitous primers for nucleic acidamplification (speA sequences). Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionBacterial species: Streptococcus pyogenes 9945′-TGG ACT AAC AAT CTC GCA AGA GG 993^(a) 60-82  995^(b)5′-ACA TTC TCG TGA GTA ACA GGG T 993^(a) 173-194 9965′-ACA AAT CAT GAA GGG AAT CAT TTA G 993^(a) 400-424  997^(b)5′-CTA ATT CTT GAG CAG TTA CCA TT 993^(a) 504-526 9985′-GGA GGG GTA ACA AAT CAT GAA GG 993^(a) 391-413  997^(b)5′-CTA ATT CTT GAG CAG TTA CCA TT 993^(a) 504-526 ^(a)Sequence fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.

TABLE 61First strategy for the selection of Streptococcus pyogenes-specificamplification primers from speA sequences. ACCESSION #57                            85   170                         197SEQ ID NO.: speA X61573CCTT GGgCTAACAA cCTCaCAAGA aGTAT...GTGAtCCT.GT cgtTCAtGAG AATGTAAA 2777speA AF029051 ~~~~GGgCTAACAA cCTCaCAAGA aGTAT...GTGAtCCT.GT cgtTCAtGAG AATGTAAA 2778 speAX61571TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2779speA X61570TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2780speA X61568TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2781speA X61569TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2782speA X61572TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2783speA X61560TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2784speA U40453TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 993speA X61554TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2785speA X61557TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2786speA X61559TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2787speA X61558TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2788speA X61556TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2789speA X61555TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2790speA X61560TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2784speA X61561TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2791speA X61566TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2792speA X61567TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2793speA X61562TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2794speA X61563TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2795speA X61564TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2796speA X61565TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2797speA AF055698 ~~~~GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2798 speAX03929^(a)TCTT GGACTAACAA TCTtGCcAaA aGGTA...GTGACCCTGGT TACTCACGAG AATGTGAA 2799Selected sequence for    T GGACTAACAA TCTCGCAAGA GG 994species-specific primer Selected sequence for                                      ACCCT.GT TACTCACGAG AATGT 995species-specific primer^(b) The sequence numbering refers to theStreptococcus pyogenes speA gene fragment (SEQ ID NO. 993). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. “~” indicateincomplete sequence data. Dots indicate gaps in the sequences displayed.^(a)The extra G nucleotide introducing a gap in the sequence is probablya sequencing error. ^(b)This sequence is the reverse-complement of theselected primer.

TABLE 62Second strategy for the selection of Streptococcus pyogenes-specificamplification primers from speA sequences. SEQ ID Accession #388                                      427   501                         529NO.: speA X61573TA TGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2777 speA AF029051TA TGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2778 speA X61571TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2779 speA X61570TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2780 speA X61568TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2781 speA X61569TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2782 speA X61572TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2783 speA X61560TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2784 speA U40453TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT993 speA X61554TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2785 speA X61557TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2786 speA X61559TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2787 speA X61558TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2788 speA X61556TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2789 speA X61555TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2790 speA X61560TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2784 speA X61561TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2791 speA X61566TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2792 speA X61567TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2793 speA X61562TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2794 speA X61563TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2795 speA X61564TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2796 speA X61565TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2797 speA AF055698TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2798 speA X03929TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAG.CT2799 Selected sequences     GGAGGGGTA ACAAATCATG AAGG 998 for              ACAAATCATG AAGGGAATCA TTTAG 996 species-specific primersSelected sequence                                                  AATGGT AACTGCTCAA GAATTAG997 for species-specific primer^(a) The sequence numbering refers to theStreptococcus pyogenes speA gene fragment (SEQ ID NO. 993). Dotsindicate gaps in the sequences displayed. ^(a)This sequence is thereverse-complement of the selected primer.

TABLE 63 Strategy for the selection of Streptococcus pyogenes-specificamplification primers from tuf sequences.140                                              186   619                          647SEQ ID NO.: S. anginosusA AGTTGACtTg GTTGACGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt cAtCCACACA CTAAATT211 S. bovisA AGTTGACCTT GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC cACCCACACA CTAAATT212 S. dysgalactiaeA AATTGACCTT GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT217 S. pyogenesA AGTTGACCTT GTTGATGACG AAGAGTTGCT TGAATTAGTT GAGATG...CC AAGTTCAATC AACCCACACA CTAAATT1002 S. agalactiaeA AGTTGACCTT GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT2860^(a) S. oralisA AATTGACtTg GTAGAcGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT985 S. pneumoniaeA AGTTGACtTg GTTGAcGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT145^(a) S. cristatusA GATCGACtTg GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT215 S. mitisA GATCGACtTg GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT982 S. gordoniiA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAGTTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT200 S. sanguinisA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT227 S. parasanguinisA AGTTGACtTg GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT225 S. salivariusA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC TgGTTCAATC AACCCACACA CTAAATT2862^(c) S. vestibularisA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG.. CC TgGTTCAATC AACCCACACA CTAAATT231 S. suisA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAgTTgGTT GAaATG...CC AgGTTCtATC AACCCACACA CTAAATT229 S. mutansA AGTTGAttTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt cACCCACAcA CTAAATT224 S. rattiA GGTTGACtTg GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt cAtCCgCAcA CTAAATT226 S. macacaeA AGTTGACtTa GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGATCAATt cAtCCACAcA CTAAATT222 S. cricetusA GGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC TgGTTCAATC cAtCCACACA CTAAATT214 E. faecalisA AATgGAtaTg GTTGATGACG AAGAaTTatT aGAATTAGTa GAaATG...CC AgcTaCAATC ActCCACACA CaAAATT607 S. aureusA AGTTGACaTg GTTGAcGAtG AAGAaTTatT aGAATTAGTa GAaATG...CC TgGTTCAATt AcaCCACACA CTgAATT176 B. cereusA ATgcGACaTg GTaGATGACG AAGAaTTatT aGAATTAGTa GAaATG...AG CgGTTCtgTa AAagCtCACg CTAAATT7 E. coliA ATgcGACaTg GTTGATGACG AAGAGcTGCT gGAAcTgGTT GAaATG...CC GgGCaCcATC AAgCCgCACA CcAAGTT78 Selected sequences for species-specific primers    TTGACCTT GTTGATGACG AAGAG 999                          AAGAGTTGCT TGAATTAGTT GAG 1001Selected sequence for species-specific primer^(b)                                                           AGTTCAATC AACCCACACA CTAA1000 The sequence numbering refers to the Streptococcus pyogenes tufgene fragment (SEQ ID NO. 1002). Nucleotides in capitals are identicalto the selected sequences or match those sequences. Mismatches areindicated by lower-case letters. Dots indicate gaps in the sequencesdisplayed. ^(a)This sequence corresponds to SEQ ID NO. 144 of previouspatent publication WO98/20157. ^(b)This sequence is thereverse-complement of the selected primer. ^(c)This sequence correspondsto SEQ ID NO. 146 of previous patent publication WO98/20157.

TABLE 64Strategy for the selection stx₁-specific amplification primers andhybridization probe. SEQ ID Accession #230                               263   343                              375   391                            421NO.: stx₁ M19473aTTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2800 stx₁ M16625TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2801 stx₁ M17358TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2802 stx₁ Z36900TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTaGTGA CAGTAGCTAT ACCA2803 stx₁ L04539TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2804 stx₁ M19437TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2805 stx₁ M24352TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2806 stx₁ X07903TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2807 stx₁ Z36899TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2808 stx₁ Z36901TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA1076 stx₂ X61283TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2809 stx₂ L11079TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2810 stx₂ M21534TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2811 stx₂ M36727TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2812 stx₂ X81415TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2813 stx₂ X81416TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2814 stx₂ X81417TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2815 stx₂ X81418TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2816 stx₂ E03962TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2817 stx₂ E03959TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2818 stx₂ X07865TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2819 stx₂ Y10775TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2820 stx₂ Z37725TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA1077 stx₂ Z50754TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2821 stx₂ X67514TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2822 stx₂ L11078TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2823 stx₂ X65949TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2824 stx₂ AF043627TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2825 Selected sequence for    ATGTC AGAGGGATAG ATCCAGAGGA AGG 1081amplification primer Selected sequence for                                           CG CTTTGCTGAT TTTTCACATG TTACC1084 hybridization probe Selected sequence for                                                                                ACAT TGTCTGGTGA CAGTAGCTAT A1080 amplification primer^(a) The sequence numbering refers to theEscherichia coli stx₁ gene fragment (SEQ ID NO. 1076). Nucleotides incapitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)This sequence is thereverse-complement of the selected primer.

TABLE 65Strategy for the selection of stx₂-specific amplification primers andhybridization probe. Accession #543                         570   614                            641   684                      708SEQ ID NO.: stx₁ M19473AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gCGTcCTgCC tGAC2852 stx₁ M16625AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gCGTcCTgCC tGAC2801 stx₁ M17358AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gCGTcCTgCC tGAC2802 stx₁ Z36900AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAT2803 stx₁ L04539AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAT2804 stx₁ M19437AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC2805 stx₁ M24352AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC2806 stx₁ X07903AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC2807 stx₁ Z36899AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC2808 stx₁ Z36901AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTtgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC1076 stx₂ X61283AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2809 stx₂ L11079AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2810 stx₂ M21534AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2811 stx₂ M36727AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2812 stx₂ U72191AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2826 stx₂ X81415AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2813 stx₂ X81416AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2814 stx₂ X81417AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2815 stx₂ X81418AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2816 stx₂ E03962AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2817 stx₂ E03959AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2818 stx₂ X07865AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2819 stx₂ Y10775AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2820 stx₂ Z37725AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG1077 stx₂ Z50754AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2821 stx₂ X67514AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2822 stx₂ L11078AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G AGAATCAGCA ATGTGCTTCC GGAG2823 stx₂ X65949AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G AGAATCAGCA ATGTGCTTCC GGAG2824 stx₂ AF043627AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2825 Selected sequence for    AG TTCTGCGTTT TGTCACTGTC 1078amplification primer Selected sequence for                                     C ACTGTCTGA. ..AACTGCTC CTGT 1085hybridization probe Selected sequence for                                                                           AATCAGCA ATGTGCTTCC G1079 amplification primer^(a) The sequence numbering refers to theEscherichia coli stx₂ gene fragment (SEQ ID NO. 1077). Nucleotides incapitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)This sequence is thereverse-complement of the selected primer.

TABLE 66Strategy for the selection of vanA-specific amplification primers fromvan sequences. Accession #926                        952   1230                     1255SEQ ID NO.: vanA X56895GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1139 vanAM97297 GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG1141 vanA —GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1051 vanA— GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1052vanA — GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG1053 vanA —GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1054 vanA— GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1055vanA — GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG1056 vanA —GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1057 vanA— GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1049vanA — GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG1050 vanB U94526GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 1117 vanBU94527 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG CtCGTGTtGA TCTT2827 vanB U94528GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2828 vanBU94529 GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT2829 vanB U94530GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2830 vanBZ83305 GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT2831 vanB U81452GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2832 vanBU35369 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG CtCGTGTtGA TCTT2833 vanB U72704GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2834 vanBL06138 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG CtCGTGTtGA TCTT2835 vanB L15304GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2836 vanBU00456 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG CtCGTGTtGA TCTT2837 vanD AF130997GTAtgc AagGCaGAaG AAcTGcAgGC A...GC AGAGGatTgG CCCGcaTtGA cCTG 2838 vanEAF136925 GTAgAa caaaaaagtG AtTTatAtAA A...GC AaAGGatTAG CgaGaaTcGA cTTT2839 Selected sequence    AAT AGCGCGGACG AATTGGAC 1090 for amplificationprimer Selected sequence                                    GAGGTCTAG CCCGTGTGGA T 1089for amplification primer^(a) The sequence numbering refers to theEnterococcus faecium vanA gene fragment (SEQ ID NO. 1139). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)This sequence is thereverse-complement of the above selected primer.

TABLE 67Strategy for the selection of vanB-specific amplification primers fromvan sequences. Accession #470                       495   608                       633SEQ ID NO.: vanA X56895A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1139 vanAM97297 A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA1141 vanA —A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1051 vanA— A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1052vanA — A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA1053 vanA —A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1054 vanA— A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1055vanA — A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA1056 vanA —A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1057 vanA— A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1049vanA — A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA1050 vanB U94526C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 1117 vanBU94527 C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2827 vanB U94528C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 2828 vanBU94529 C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2829 vanB U94530C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 2830 vanBZ83305 C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2831 vanB U81452C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 2832 vanBU35369 C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2833 vanB U72704C TGCGATAGAA GCgGCAGGAC AATAT...ATG GTATCTTCCG CATCCATCAG GAA 2834 vanBL06138 C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2835 vanB L15304C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 2836 vanBU00456 C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2837 vanD AF130997C AGCaATcGAA GaAGCAaGAa AATAT...ACG GctTtTTtaa gATtCATCAG GAA 2838 vanEAF136925 A AGCaATAGAc GaAGCttcAa AATAT...ATG GctTtTTCga CtatgAagAG AAA2839 Selected sequence     CGATAGAA GCAGCAGGAC AA 1095 for amplificationprimer Selected sequence                                    GTATCTTCCG CATCCATCAG 1096for amplification primer^(a) The sequence numbering refers to theEnterococcus faecium vanB gene fragment (SEQ ID NO. 1117). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)This sequence is thereverse-complement of the above vanB sequence.

TABLE 68Strategy for the selection of vanC-specific amplification primers from vanCsequences. Accession #929                          957   1064                         1092SEQ ID NO.: vanC1 —GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT 1058vanC1 —GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT 1059vanC1 M75132GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT 1138vanC2 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1060vanC2 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1061vanC2 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1062vanC2 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1063vanC2 L29638GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 2840vanC2 L29638GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 2840vanC3 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1064vanC3 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1065vanC3 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGaC TTGCTCGCAT CGACTTTTTT GT 1066vanC3 L29639GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 2853Selected sequence     GACGGYTTT TTYGATTTTG AAGA 1101 for resistanceprimer Selected sequence                                      GGTC TKGCTCGMAT CGAYTTTTT 1102for resistance primer^(a) The sequence numbering refers to the vanC1gene fragment (SEQ ID NO. 1138). Nucleotides in capitals are identicalto the selected sequences or match those sequences. Mismatches areindicated by lower-case letters. Dots indicate gaps in the sequencedisplayed. “R” “Y” “M” “K” “W” and “S” designate nucleotide positionswhich are degenerated. “R” stands for A or G; “Y” stands for C or T; “M”stands for A or C; “K” stands for G or T; “W” stands for A or T; “S”stands for C or G. “I” stands for inosine which is a nucleotide analogthat can bind to any of the four nucleotides A, C, G or T. ^(a)Thissequence is the reverse-complement of the selected sequence.

TABLE 69 Strategy for the selection of Streptococcus pneumoniae-specificamplification primers and hybridization probes from pbp1a sequences.Accession #453                                                     505   678                          706SEQ ID NO.: pbp1a M90528A TTGACTAcCC AAGCATaCAc TATGCtAAtG CtATTTCAAG TAATACAACC GA...TATATG ATGACaGAtA TGATGAAAAC CGT...2841 pbp1a X67873A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGAAA TGATGAAAAC AGT...2842 pbp1a AB006868A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGACA TGATGAAAAC AGT...2843 pbp1a AF046234A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGAAA TGATGAAAAC TGT...2844 pbp1aA TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGACA TGATGAAAAC TGT...1014 pbp1aA TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1017 pbp1a AB006873A TCGACTAcCC AAGtcTtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGACA TGATGAAAAC AGT...2845 pbp1a AF139883A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1169 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1004 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1007 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1008 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1009 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1011 pbp1a AF159448A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...2846 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1005 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1015 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1006 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1012 pbp1a X67867A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...2847 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...1010 pbp1a Z49094A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...2848 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...1013 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...1016 pbp1a X67870A TCGACTATCC AAGtATGCAT TAcGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACCGAAA TGATGAAAAC TGT...2849 pbp1aA TTGACTATCC AAGtATtCAc TActCAAAtG CtATTTCAAG TAATACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...1018 pbp1a AJ002290A TTGAtTAcCC AActATGgtc TATGCtAACG CtATTTCAAG TAATACAACT GA...TACATG ATGACtGAAA TGATGAAAAC AGT...2850 pbp1a X67871A TCGACTAcCC AAGtcTtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TACATG ATGACaGAAA TGATGAAAAC AGT...2851 Selected sequences for     GACTATCC AAGCATGCAT TATG 1130amplification primers                                                                 ATG ATGACHGAMA TGATGAAAAC1129 Selected sequence for                            CAAACG CCATTTCAAG TAATACAAC 1197hybridization probeThe sequence numbering refers to the Streptococcus pneumoniae pbp1a gene fragment (SEQ ID NO. 1004). Nucleotides in capitals are identical to the selected sequences or match those sequences.Mismatches are indicated by lower-case letters. Dotes indicate gaps in the sequences displayed.“R” “Y” “M” “K” “W” and “S”designate nucleotide positions which are degenerated. “R” stands forA or G; “Y” stands for C or T; “M” stands for A or C; “K”stands for G or T; “W” stands for A or T; “H” stands for A, C or T; “S”stands for C or G. “I”stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.Accession #   756                        783   813                         840SEQ ID NO.: pbp1a M90528...GCTGGTAA aACtGGTACg TCTaACTATA...A ATACgGGTTA TGTAGCTCCG GAcGAAA 2841pbp1a X67873...GCTGGTAA aACAGGaACc TCTAACTATA...A CCtCTcaaTt TGTAGCaCCt GATGAAC 2842pbp1a AB006868...GCTGGTAA aACAGGaACc TCTAACTATA...A CCtCTcaaTt TGTAGCaCCt GAcGAAC 2843pbp1a AF046234...GCAGGTAA aACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 2844pbp1a...GCAGGTAA aACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 1014pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1017pbp1a AB006873...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 2845pbp1a AF139883...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1169pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1004pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1007pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1008pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1009pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1011pbp1a AF159448...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 2846pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1005pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1015pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1006pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1012pbp1a X67867...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 2847pbp1a...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 1010pbp1a Z49094...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 2848pbp1a...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 1013pbp1a...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 1016pbp1a X67870...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 2849pbp1a...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 1018pbp1a AJ002290...GCAGGTAA GACgGGTACa TCTAACTACA...A ACACTGGCTA C~~~~~~~~~ ~~~~~~~ 2850pbp1a X67871...GCTGGTAA aACAGGTACc TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 2851Selected sequence for       GGTAA GACAGGTACT TCTAACT 1193hybridization probe Selected sequence for                                        ACTGGYTA YGTAGCTCCA GATG 1131amplification primer^(a)The sequence numbering refers to the Streptococcus pneumoniae pbp1a gene fragment (SEQ ID NO. 1004). Nucleotides in capitals are identical to the selected sequences or match those sequences.Mismatches are indicated by lower-case letters. Dots indicate gaps in the sequences displayed. “~”indicates incomplete sequence data. “R” “Y” “W” and “S”designate nucleotide positions which are degenerated. “R”stands for A or G; “Y” stands for C or T; “W” stands for A or T; “S”stands for C or G. “I”stands for inosine which is a nucleotide analog that can bind to any of thefour nucleotides A, C, G or T.^(a)This sequence is the reverse-complement of the selected primer.

TABLE 70 Specific and ubiquitous primers for nucleic acidamplification (toxin sequences). Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. position Toxin gene: cdtA2123 5′-TCT ACC ACT GAA GCA TTA C 2129^(a) 442-460  2124^(b)5′-TAG GTA CTG TAG GTT TAT TG 2129^(a) 580-599 Toxin gene: cdtB 21265′-ATA TCA GAG ACT GAT GAG 2130^(a) 2665-2682  2127^(b)5′-TAG CAT ATT CAG AGA ATA TTG T 2130^(a) 2746-2767 Toxin gene: stx₁1081 5′-ATG TCA GAG GGA TAG ATC CA 1076^(a) 233-252  1080^(b)5′-TAT AGC TAC TGT CAC CAG ACA ATG T 1076^(a) 394-418 Toxin gene: stx₂1078 5′-AGT TCT GCG TTT TGT CAC TGT C 1077^(a) 546-567  1079^(b)5′-CGG AAG CAC ATT GCT GAT T 1077^(a) 687-705 Toxin genes: stx₁ and stx₂1082 5′-TTG ARC RAA ATA ATT TAT ATG TG 1076^(a) 278-300  1083^(b)5′-TGA TGA TGR CAA TTC AGT AT 1076^(a) 781-800 ^(a)Sequences fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.

TABLE 71 Molecular beacon internal hybridization probes forspecific detection of toxin sequences. Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence^(a) NO. positionToxin gene: cdtA 2125^(b) 5′-CAC GCG GAT TTT GAA TCT CTT CCT CTA2129^(c) 462-488 GTA GCG CGT G Toxin gene: cdtB 21285′-CAA CGC TGG AGA ATC TAT ATT TGT AGA 2130^(c) 2714-2740 AAC TGC GTT GToxin gene: stx₁ 1084 5′-CCA CGC CGC TTT GCT GAT TTT TCA CAT 1076^(c)337-363 GTT ACC GCG TGG 2012^(d) 5′-CCG CGG ATT ATT AAA CCG CCC TTC CGC1076^(c) 248-264 GG-MR-HEG-ATG TCA GAG GGA TAG ATC CA Toxin gene: stx₂1085 5′-CCA CGC CAC TGT CTG AAA CTG CTC CTG 1077^(c) 617-638 TG CGT GG^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)These sequences are from the complementary DNA strand of thesequence of the originating fragment given in the Sequence Listing.^(c)Sequences from databases. ^(d)Scorpion primer.

TABLE 72 Specific and ubiquitous primers for nucleic acidamplification (van sequences). Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionResistance gene: vanA 1086 5′-CTA CTC CCG CCT TTT GGG TT 1049-1057^(a)513-532^(b) 1087^(c) 5′-CTC ACA GCC CGA AAC AGC CT 1049-1057^(a)699-718^(b) 1086 5′-CTA CTC CCG CCT TTT GGG TT 1049-1057^(a) 513-532^(b)1088^(c) 5′-TGC CGT TTC CTG TAT CCG TC 1049-1057^(a) 885-904^(b) 10865′-CTA CTC CCG CCT TTT GGG TT 1049-1057^(a) 513-532^(b) 1089^(c)5′-ATC CAC ACG GGC TAG ACC TC 1049-1057^(a) 933-952^(b) 10905′-AAT AGC GCG GAC GAA TTG GAC 1049-1057^(a) 629-649^(b) 1091^(c)5′-AAC GCG GCA CTG TTT CCC AA 1049-1057^(a) 734-753^(b) 10905′-AAT AGC GCG GAC GAA TTG GAC 1049-1057^(a) 629-649^(b) 1089^(c)5′-ATC CAC ACG GGC TAG ACC TC 1049-1057^(a) 933-952^(b) 10925′-TCG GCA AGA CAA TAT GAC AGC 1049-1057^(a) 662-682^(b) 1088^(c)5′-TGC CGT TTC CTG TAT CCG TC 1049-1057^(a) 885-904^(b)Resistance gene: vanB 1095 5′-CGA TAG AAG CAG CAG GAC AA 1117^(d)473-492  1096^(c) 5′-CTG ATG GAT GCG GAA GAT AC 1117^(d) 611-630 Resistance genes: vanA, vanB 1112 5′-GGC TGY GAT ATT CAA AGC TC1049-1057, 1117^(a) 437-456^(b) 1113^(c) 5′-ACC GAC CTC ACA GCC CGA AA1049-1057, 1117^(a) 705-724^(b) 1112 5′-GGC TGY GAT ATT CAA AGC TC1049-1057, 1117^(a) 437-456^(b) 1114^(c) 5′-TCW GAG CCT TTT TCC GGC TCG1049-1057, 1117^(a) 817-837^(b) 11155′-TTT CGG GCT GTG AGG TCG GBT GHG CG 1049-1057, 1117^(a) 705-730^(b)1114^(c) 5′-TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a) 817-837^(b)1116 5′-TTT CGG GCT GTG AGG TCG GBT GHG CGG 1049-1057, 1117^(a)705-731^(b) 1114^(c) 5′-TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a)817-837^(b) 1112 5′-GGC TGY GAT ATT CAA AGC TC 1049-1057, 1117^(a)437-456^(b) 1118^(c) 5′-TTT TCW GAG CCT TTT TCC GGC TCG1049-1057, 1117^(a) 817-840^(b)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the vanA sequence fragment (SEQ ID NO.1051).cThese sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)Sequences from databases. 1115 5′-TTT CGG GCT GTG AGG TCG GBT GHG CG1049-1057, 1117^(a) 705-730^(b) 1118^(c)5′-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a) 817-840^(b) 11165′-TTT CGG GCT GTG AGG TCG GBT GHG CGG 1049-1057, 1117^(a) 705-731^(b)1118^(c) 5′-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a)817-840^(b) 1119 5′-TTT CGG GCT GTG AGG TCG GBT GHG C1049-1057, 1117^(a) 705-729^(b) 1118^(c)5′-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a) 817-840^(b) 11205′-TTT CGG GCT GTG AGG TCG GBT GHG 1049-1057, 1117^(a) 705-728^(b)1118^(c) 5′-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a)817-840^(b) 1121 5′-TGT TTG WAT TGT CYG GYA TCC C 1049-1057, 1117^(a)408-429^(b) 1111^(c) 5′-CTT TTT CCG GCT CGW YTT CCT GAT G1049-1057, 1117^(a) 806-830^(b) 1112 5′-GGC TGY GAT ATT CAA AGC TC1049-1057, 1117^(a) 437-456^(b) 1111^(c)5′-CTT TTT CCG GCT CGW YTT CCT GAT G 1049-1057, 1117^(a) 806-830^(b)1123 5′-TTT CGG GCT GTG AGG TCG GBT G 1049-1057, 1117^(a) 705-726^(b)1111^(c) 5′-CTT TTT CCG GCT CGW YTT CCT GAT G 1049-1057, 1117^(a)806-830^(b) 1112 5′-GGC TGY GAT ATT CAA AGC TC 1049-1057, 1117^(a)437-456^(b) 1124^(c) 5′-GAT TTG RTC CAC YTC GCC RAC A1049-1057, 1117^(a) 757-778^(b) Resistance gene: vanC1 11035′-ATC CCG CTA TGA AAA CGA TC 1058-1059^(a) 519-538^(d) 1104^(c)5′-GGA TCA ACA CAG TAG AAC CG 1058-1059^(a) 678-697^(d)Resistance genes: vanC1, vanC2, vanC3 10975′-TCY TCA AAA GGG ATC ACW AAA GTM AC 1058-1066^(a) 607-632^(d) 1098^(c)5′-TCT TCA AAA TCG AAA AAG CCG TC 1058-1066^(a) 787-809^(d) 10995′-TCA AAA GGG ATC ACW AAA GTM AC 1058-1066^(a) 610-632^(d) 1100^(c)5′-GTA AAK CCC GGC ATR GTR TTG ATT TC 1058-1066^(a) 976-1001^(d) 11015′-GAC GGY TTT TTY GAT TTT GAA GA 1058-1066^(a) 787-809^(d) 1102^(c)5′-AAA AAR TCG ATK CGA GCM AGA CC 1058-1066^(a) 922-944^(d)Resistance genes: vanC2, vanC3 1105 5′-CTC CTA CGA TTC TCT TGA YAA ATC A1060-1066, 1140^(a) 487-511^(e) 1106^(c)5′-CAA CCG ATC TCA ACA CCG GCA AT 1060-1066, 1140^(a) 690-712^(e)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the vanA sequence fragment (SEQ ID NO.1051).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the vanC1 sequence fragment (SEQ ID NO.1058).^(e)The nucleotide positions refer to the vanC2 sequence fragment (SEQ ID NO.1140). Resistance gene: vanD 1591 5′-ATG AGG TAA TAG AAC GGA TT 1594797-837  1592^(b) 5′-CAG TAT TTC AGT AAG CGT AAA 1594 979-999 Resistance gene: vanE 1595 5′-AAA TAA TGC TCC ATC AAT TTG CTG A 1599^(a)74-98  1596^(b) 5′-ATA GTC GAA AAA GCC ATC CAC AAG 1599^(a) 394-417 1597 5′-GAT GAA TTT GCG AAA ATA CAT GGA 1599^(a) 163-186  1598^(b)5′-CAG CCA ATT TCT ACC CCT TTC AC 1599^(a) 319-341 Sequencing primers (vanAB) 1112 5′-GGC TGY GAT ATT CAA AGC TC 1139^(a)737-756  1111^(b) 5′-CTT TTT CCG GCT CGW YTT CCT GAT G 1139^(a)1106-1130  Sequencing primers (vanA, vanX, vanY) 11505′-TGA TAA TCA CAC CGC ATA CG 1141^(a) 860-879  1151^(b)5′-TGC TGT CAT ATT GTC TTG CC 1141^(a) 1549-1568  11525′-ATA AAG ATG ATA GGC CGG TG 1141^(a) 1422-1441  1153^(b)5′-CTC GTA TGT CCC TAC AAT GC 1141^(a) 2114-2133  11545′-GTT TGA AGC ATA TAG CCT CG 1141^(a) 2520-2539  1155^(b)5′-CAG TGC TTC ATT AAC GTA GTC 1141^(a) 3089-3109 Sequencing primers (vanC1) 1110 5′-ACG AGA AAG ACA ACA GGA AGA CC1138^(a) 122-144  1109^(b) 5′-ACA TCG TGA TCG CTA AAA GGA GC 1138^(a)1315-1337  Sequencing primers (vanC2, vanC3) 11085′-GTA AGA ATC GGA AAA GCG GAA GG 1140^(a) 1-23 1107^(b)5′-CTC ATT TGA CTT CCT CCT TTG CT 1140^(a) 1064-1086 ^(a)Sequences from databases.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.

TABLE 73 Internal hybridization probes forspecific detection of van sequences. Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionResistance gene: vanA 1170 5′-ACG AAT TGG ACT ACG CAA TT 1049-1057^(a) 639-658^(b) 2292 5′-GAA TCG GCA AGA CAA TAT G 2293^(c) 583-601Resistance gene: vanB 1171 5′-ACG AGG ATG ATT TGA TTG TC 1117^(c)560-579 2294 5′-AAA CGA GGA TGA TTT GAT TG 2296^(a) 660-679 22955′-TTG AGC AAG CGA TTT CGG 2296^(a) 614-631 Resistance gene: vanD 22975′-TTC AGG AGG GGG ATC GC 1594^(c) 458-474 ^(a)These sequences werealigned to derive the corresponding primer. ^(b)The nucleotide positionsrefer to the vanA sequence fragment (SEQ ID NO. 1051). ^(c)Sequencesfrom databases.

TABLE 74 Specific and ubiquitous primers for nucleic acidamplification (pbp sequences). Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionResistance gene: pbp1a 1129 5′-ATG ATG ACH GAM ATG ATG AAA AC1004-1018^(a)  681-703^(b) 1131^(c) 5′-CAT CTG GAG CTA CRT ARC CAG T1004-1018^(a)  816-837^(b) 1130 5′-GAC TAT CCA AGC ATG CAT TAT G1004-1018^(a)  456-477^(b) 1131 5′-CAT CTG GAG CTA CRT ARC CAG T1004-1018^(a)  816-837^(b) 2015 5′-CCA AGA AGC TCA AAA ACA TCT G2047^(d) 909-930 2016^(c) 5′-TAD CCT GTC CAW ACA GCC AT 2047^(d)1777-1796 Sequencing primers (pbp1a) 1125 5′-ACT CAC AAC TGG GAT GGA TG1169^(d) 873-892 1126^(c) 5′-TTA TGG TTG TGC TGG TTG AGG 1169^(d)2140-2160 1125 5′-ACT CAC AAC TGG GAT GGA TG 1169^(d) 873-892 1128^(c)5′-GAC GAC YTT ATK GAT ATA CA 1169^(d) 1499-1518 11275′-KCA AAY GCC ATT TCA AGT AA 1169^(d) 1384-1403 1126^(c)5′-TTA TGG TTG TGC TGG TTG AGG 1169^(d) 2140-2160Sequencing primers (pbp2b) 1142 5′-GAT CCT CTA AAT GAT TCT CAG GTG G1172^(d)  1-25 1143^(c) 5′-CAA TTA GCT TAG CAA TAG GTG TTG G 1172^(d)1481-1505 1142 5′-GAT CCT CTA AAT GAT TCT CAG GTG G 1172^(d)  1-251145^(c) 5′-AAC ATA TTK GGT TGA TAG GT 1172^(d) 793-812 11445′-TGT YTT CCA AGG TTC AGC TC 1172^(d) 657-676 1143^(c)5′-CAA TTA GCT TAG CAA TAG GTG TTG G 1172^(d) 1481-1505Sequencing primers (pbp2x) 1146 5′-GGG ATT ACC TAT GCC AAT ATG AT1173^(d) 219-241 1147^(c) 5′-AGC TGT GTT AGC VCG AAC ATC TTG 1173^(d)1938-1961 1146 5′-GGG ATT ACC TAT GCC AAT ATG AT 1173^(d) 219-2411149^(c) 5′-TCC YAC WAT TTC TTT TTG WG 1173^(d) 1231-1250 11485′-GAC TTT GTT TGG CGT GAT AT 1173^(d) 711-730 1147^(c)5′-AGC TGT GTT AGC VCG AAC ATC TTG 1173^(d) 1938-1961 ^(a)Thesesequences were aligned to derive the corresponding primer. ^(b)Thenucleotide positions refer to the pbp1a sequence fragment (SEQ ID NO.1004). ^(c)These sequences are from the complementary DNA strand of thesequence of the originating fragment given in the Sequence Listing.^(d)Sequences from databases.

TABLE 75 Internal hybridization probes for specific detectionof pbp sequences. Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Resistance gene: pbp1a 11325′-AGT GAA AAR ATG GCT GCT GC 1004-1018^(a)  531-550^(b) 11335′-CAT CAA GAA CAC TGG CTA YGT AG 1004-1018^(a)  806-828^(b) 11345′-CTA GAT AGA GCT AAA ACC TTC CT 1004-1018^(a)  417-439^(b) 11355′-CAT TAT GCA AAC GCC ATT TCA AG 1004-1018^(a)  471-493^(b) 11925′-GGT AAA ACA GGA ACC TCT AAC T 1004-1018^(a)  759-780^(b) 11935′-GGT AAG ACA GGT ACT TCT AAC T 1004-1018^(a)  759-780^(b) 11945′-CAT TTC AAG TAA TAC AAC AGA ATC 1004-1018^(a)  485-508^(b) 11955′-CAT TTC AAG TAA CAC AAC TGA ATC 1004-1018^(a)  485-508^(b) 11965′-GCC ATT TCA AGT AAT ACA ACA GAA 1004-1018^(a)  483-506^(b) 11975′-CAA ACG CCA TTT CAA GTA ATA CAA C 1004-1018^(a)  478-502^(b) 10945′-GGT AAA ACA GGT ACT TCT AAC TA 1004-1018^(a)  759-781^(b) 12145′-GGT AAA ACA GGT ACC TCT AAC TA 1004-1018^(a)  759-781^(b) 12165′-GGT AAG ACT GGT ACA TCA AAC TA 1004-1018^(a)  759-781^(b) 12175′-CAA ATG CCA TTT CAA GTA ACA CAA C 1004-1018^(a)  478-502^(b) 12185′-CAA ACG CCA TTT CAA GTA ACA CAA C 1004-1018^(a)  478-502^(b) 12195′-CAA ATG CTA TTT CAA GTA ATA CAA C 1004-1018^(a)  478-502^(b) 12205′-CAA ACG CCA TTT CAA GTA ATA CGA C 1004-1018^(a)  478-502^(b) 20175′-ACT TTG AAT AAG GTC GGT CTA G 2047^(c) 1306-1327 20185′-ACA CTA AAC AAG GTT GGT TTA G 2063 354-375 20195′-ACA CTA AAC AAG GTC GGT CTA G 2064 346-367 20205′-GTA GCT CCA GAT GAA ATG TTT G 2140^(c) 1732-1753 20215′-GTA GCT CCA GAC GAA ATG TTT G 2057 831-852 20225′-GTA GCT CCA GAT GAA ACG TTT G 2053^(c) 805-826 20235′-GTA ACT CCA GAT GAA ATG TTT G 2056 819-840 20245′-AGT GAA AAG ATG GCT GCT GC 2048^(c) 1438-1457 20255′-AGT GAG AAA ATG GCT GCT GC 2047^(c) 1438-1457 20265′-TCC AAG CAT GCA TTA TGC AAA CG 2047^(c) 1368-1390 20275′-TCG GTC TAG ATA GAG CTA AAA CG 2047^(c) 1319-1341 20285′-TAT GCT CTT CAA CAA TCA CG 2047^(c) 1267-1286 20295′-AGC CGT TGA GAC TTT GAA TAA G 2047^(c) 1296-1317 20305′-CTT AAT GGT CTT GGT ATC G 2047^(c) 1345-1366 20315′-CGT GAC TGG GGT TCT GCT ATG A 2049^(c) 1096-1117 20325′-CGT GAC TGG GGA TCA TCA ATG A 2047^(c) 1096-1117 20335′-CGT GAC TGG GGT TCT GCC ATG A 2057 195-216 20345′-ATC AAG AAC ACT GGC TAT GTA G 2050^(c) 787-808 20355′-ATC AAG AAC ACT GGC TAC GTA G 2051^(c) 787-808 20365′-ATC AAG AAC ACT GGT TAC GTA G 2047 1714-1735 20375′-ATC AAA AAT ACT GGT TAT GTA G 2057 813-834 20385′-ATC AAG AAT ACT GGC TAC GTA G 2052^(c) 757-778 20395′-ATC AAA AAC ACT GGC TAT GTA G 2053^(c) 787-808 ^(a)These sequenceswere aligned to derive the corresponding primer. ^(b)The nucleotidepositions refer to the pbp1a sequence fragment (SEQ ID NO. 1004).^(c)Sequence from databases.

TABLE 76Strategy for the selection of vanAB-specific amplification primers andvanA- and vanB-specific hybridization probes from van sequences.Accession # 734                      759   936                      961SEQ ID NO.: vanA X56895GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA... 1139 vanAM97297 GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1141 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1051 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1052 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1053 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1054 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1055 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1056 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1057 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1049 vanA GTAGGCT GCGATATTCA AAGCTCAGC...CGGACGAATT GGACTACGCA ATTGAA...1050 vanB U94526GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... 1117 vanBU94527 GTAGGCT GCGATATTCA AAGCTCCGC...CGGAaGAAcT aaACgctGCg ATaGAA...2827 vanB U94528GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... 2828 vanBU94529 GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA...2829 vanB U94530GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... 2830 vanBZ83305 GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA...2831 vanB U81452GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... 2832 vanBU35369 GTAGGCT GCGATATTCA AAGCTCCGC...CGGAaGAAcT aaACgctGCg ATaGAA...2833 vanB U72704GTGGGCT GCGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... 2834 vanBL06138 GTAGGCT GCGATATTCA AAGCTCCGC...CGGAaGAAcT aaACgctGCg ATaGAA...2835 vanB L15304GTGGGCT GTGATATTCA AAGCTCCGC...CGGAaGAAcT taACgctGCg ATaGAA... 2836 vanBU00456 GTAGGCT GCGATATTCA AAGCTCCGC...CGGAaGAAcT aaACgctGCg ATaGAA...2837 vanD AF130997GTGGGaT GCGATATTCA AAGCTCCGT...CAGAaGAAcT GcAggcaGCA ATcGAA... 2838 vanEAF136925 GTAGGtT GTGgTATcgg AgctgCAGC...AAAgtGAtTT atAtaAaGCA ATaGAC...2839 Selected    GGCT GYGATATTCA AAGCTC 1112 sequence for amplificationprimer Selected                                  ACGAATT GGACTACGCA ATT (vanA) 1170sequence for hybridization probeThe sequence numbering refers to the Enterococcus faecium vanA gene fragment (SEQ ID NO. 1139).Nucleotides in capitals are identical to the selected sequences or match those sequences.Mismatches are indicated by lower-case letters. Dots indicate gaps in the sequences displayed.“R” “Y” “M” “K” “W” and “S”designate nucleotide positions which are degenerated. “R” stands forA or G; “Y” stands for C or T; “M” stands for A or C; “K”stands for G or T; “W” stands for A or T; “S” stands for C or G. “I”stands for inosine which is a nucleotide analog that can bind to any ofthe four nucleotides A, C, G or T. Accession #1038                    1063   1103                          1133SEQ ID NO.: vanA X56895GAAACagt GccGcGTTag TTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1139vanA M97297GAAACagt GccGcgTTag TTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1141vanA GAAACagt GccGcgTTag TTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT1051 vanAGAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1052vanA GAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT1053 vanAGAAACagt GccGcgTTag TTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1054vanA GAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT1055 vanAGAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1056vanA GAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT1057 vanAGAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1049vanA GAAACagt GccGcgTTag cTGTtGGC...ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT1050 vanB U94526GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 1117vanB U94527GAAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2827vanB U94528GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2828vanB U94529GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2829vanB U94530GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2830vanB Z83305GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2831vanB U81452GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2832vanB U35369GAAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2833vanB U72704GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGAT 2834vanB L06138GAAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2835vanB L15304GGAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2836vanB U00456GAAACGAG GATGATTTGA TTGTCGGC...ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 2837vanD AF130997GAAACGga aATGATcTcA TgGctGGC...ATT CATCAGGAAG cacAGCCGGA aAAGGGAT 2838vanE AF136925GGAA...t GAacAaTTGg TcGTtGGA...TAT gAagAGaAAt ACaA...... ......TT 2839Selected    ACGAG GATGATTTGA TTGTC (vanB) 1171 sequence forhybridization probe Selected                                   CATCAGGAAR WCGAGCCGGA AAAAG 1111sequence for amplification primer^(a)The sequence numbering refers to the Enterococcus faecium vanA gene fragment (SEQ ID NO. 1139).Nucleotides in capitals are identical to the selected sequences or match those sequences.Mismatches are indicated by lower-case letters. Dots indicate gaps in the sequences displayed.“R” and “W” designate nucleotide positions which are degenerated. “R”stands for A or G; “W” stands for A or T^(a)This sequence is the reverse-complement of the above selected primer.

TABLE 77 Internal hybridization probe for specific detection of mecA.Originating DNA fragment SEQ ID SEQ ID Nucleotide NO.Nucleotide sequence NO. position Resistance gene: mecA 11775′-GCT CAA CAA GTT CCA 1178^(a) 1313-1332 GAT TA ^(a)Sequence fromdatabases.

TABLE 78 Specific and ubiquitous primers for nucleic acid amplification(hexA sequences). Originating DNA fragment SEQ ID SEQ ID Nucleotide NO.Nucleotide sequence NO. positionBacterial species: Streptococcus pneumoniae 1179 5′-ATT TGG TGA CGG1183^(a) 431-450 GTG ACT TT  1181^(b) 5′-AGC AGC TTA CTA 1183-1191^(c) 652-671^(d) GAT GCC GT Sequencing primers 1179 5′-ATT TGG TGA CGG1183^(a) 431-450 GTG ACT TT  1182^(b) 5′-AAC TGC AAG AGA 1183^(a)1045-1064 TCC TTT GG ^(a)Sequences from databases. ^(b)These sequencesare from the complementary DNA strand of the sequence of the originatingfragment given in the Sequence Listing. ^(c)These sequences were alignedto derive the corresponding primer. ^(d)The nucleotide positions referto the hexA sequence fragment (SEQ ID NO. 1183).

TABLE 79 Internal hybridization probe forspecific detection of hexA sequences. Originating DNA fragment SEQ IDSEQ ID Nucleotide NO. Nucleotide sequence NO. positionBacterial species: Streptococcus pneumoniae 1180^(a) 5′-TCC ACC GTT GCC1183-1191^(b) 629-647^(c) AAT CGC A ^(a)This sequences is from thecomplementary DNA strand of the sequence of the originating fragmentgiven in the Sequence Listing. ^(b)These sequences were aligned toderive the corresponding primer. ^(c)The nucleotide positions refer tothe hexA sequence fragment (SEQ ID NO. 1183).

TABLE 80Strategy for the selection of Streptococcus pneumoniae species-specificamplification primers and hybridization probe from hexA sequences.SEQ ID428                       453   626                                                674   1042                    1067NO.: S. pneumoniaeTGG ATTTGGTGAC GGGTGACTTT TAT...ATTTG CGATTGGCAA CGGTGGAGCA AACGGCATCT AGTAAGCTGC TCCA...AATCCAAAG GATCTCTTGC AGTTGGC1183 S. pneumoniae ~~~~~~~~~TGAC GGGTGACTTT TAT...ATTTG CGATTGGCAA CGGTGGAGCA AACGGCATCT AGTAAGCTGC TCCA...AATCCAAAG GATCTCTTG~ ~~~~~~~1184 S. pneumoniae ~~~~~~~~~TGAC GGGTGACTTT TAT...ATTTG CGATTGGCAA CGGTGGAGCA AACGGCATCT AGTAAGCTGC TCCA...AATCCAAAG GATCTCT~~~ ~~~~~~~1185 S. pneumoniae ~~~~~~~~~TGAC GGGTGACTTT TAT...ATTTG CGATTGGCAA CGGTGGAGCA AACGGCATCT AGTAAGCTGC TCCA...AATCCAAAG GATCTCTT~~ ~~~~~~~1186 S. pneumoniae ~~~~~~~~~TGAC GGGTGACTTT TAT...ATTTG CGATTGGCAA CGGTGGAGCA AACGGCATCT AGTAAGCTGC TCCG...AATCCAAAG GATCTCTT~~ ~~~~~~~1187 S. oralis ~~~~~~~~~~~~~ GGGTGACTTT TAT...ATCca CGAcTGGCAg CtGTGGAGCA AgCGGCAgCT AGTAAGCTcC TCCA...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~1188 S. mitis ~~~~~~~GGTGAC GGGTGACTTT TAT...ATTca CGATTGGCAg CtGTGGAGCA AgCGGCATCT AGTAAaCTGC TTCA...AATCCAAAG GATCTCTT~~ ~~~~~~~1189 S. mitis ~~~

~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ 1190 S. mitis ~~~~~~~~~TGAC GGGTGACTTT CAG...GCGaG gaAcTGtCtc CtaTGGAGCG TcaGGCAgCg gGgAAatTGC TAGA...AATCCAAAG GATCTCTT~~ ~~~~~~~1191 Selected sequence for     ATTTGGTGAC GGGTGACTTT 1179amplification primer Selected sequences for                                                            ACGGCATCT AGTAAGCTGC T1181 amplification primers^(a)                                                                                             CCAAAG GATCTCTTGC AGTT1182 Selected sequence for                                   TG CGATTGGCAA CGGTGGA 1180hybridization probe^(a) The sequence numbering refers to theStreptococcus pneumoniae hexA gene fragment (SEQ ID NO. 1183).Nucleotides in capitals are identical to the selected sequences or matchthose sequences. Mismatches are indicated by lower-case letters. Dotsindicate gaps in the sequences displayed. “~” indicate incompletesequence data. ^(a)This sequence is the reverse-complement of theselected primer.

TABLE 81 Specific and ubiquitous primers for nucleic acidamplification (pcp sequence). Originating DNA fragment SEQ ID NucleotideSEQ ID NO. Nucleotide sequence NO. positionBacterial species: Streptococcus pyogenes 12115′-ATT CTT GTA ACA GGC TTT GAT CCC 1215^(a) 291-314 1210^(b)5′-ACC AGC TTG CCC AAT ACA AAG G 1215^(a) 473-494 ^(a)Sequences fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.

TABLE 82 Specific and ubiquitous primers for nucleic acidamplification of S. saprophyticus sequences of unknown coding potential.Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. positionBacterial species: Staphylococcus saprophyticus 12085′-TCA AAA AGT TTT CTA AAA AAT TTA C 74, 1093, 169-193^(c) 1198^(b)1209^(a) 5′-ACG GGC GTC CAC AAA ATC AAT AGG A 74, 1093, 355-379^(c)1198^(b) ^(a)This sequence is from the complementary DNA strand of thesequence of the originating fragment given in the Sequence Listing.^(b)These sequences were aligned to derive the corresponding primer.^(c)The nucleotide positions refer to the S. saprophyticus unknown genesequence fragment (SEQ ID NO. 1198).

TABLE 83 Molecular beacon internal hybridization probes forspecific detection of antimicrobial agents resistance gene sequences.Originating DNA fragment SEQ ID SEQ ID Nucleotide NO.Nucleotide sequence^(a) NO. position Resistance gene: gyrA 22505′-CCG TCG GAT GGT GTC GTA TAC CGC GGA GTC 1954^(b) 218-243 GCC GAC GG2251 5′-CGG AGC CGT TCT CGC TGC GTT ACA TGC TGG 1954^(b) 259-286TGG CTC CG Resistance gene: mecA 12315′-GCG AGC CCG AAG ATA AAA AAG AAC CTC TGC 1178^(b) 1291-1315 TGC TCG CResistance gene: parC 1938^(b)5′-CCG CGC ACC ATT GCT TCG TAC ACT GAG GAG 1321^(c) 232-260TCT CCG CGC GG 1939 5′-CGA CCC GGA TGG TAG TAT CGA TAA TGA TCC 1321^(c)317-346 GCC AGC GGC CGG GTC G 1955^(b)5′-CGC GCA ACC ATT GCT TCG TAC ACT GAG GAG 1321^(c) 235-260 TCT GCG CGResistance gene: vanA 1239 5′-GCG AGC GCA GAC CTT TCA GCA GAG GAG GCT1051 860-880 CGC 1240 5′-GCG AGC CGG CAA GAC AAT ATG ACA GCA AAA 1051663-688 TCG CTC GC Resistance gene: vanB 12415′-GCG AGC GGG GAA CGA GGA TGA TTT GAT TGG 1117 555-577 CTC GCResistance gene: vanD 1593 5′-CCG AGC GAT TTA CCG GAT ACT TGG CTG ICG1594 835-845 CTC GG ^(a)Underlined nucleotides indicate the molecularbeacon's stem. ^(b)This sequence is from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.^(c)Sequence from databases.

TABLE 84 Molecular beacon internal hybridization probe forspecific detection of S. aureus gene sequences ofunknown coding potential. Originating DNA fragment SEQ ID SEQ IDNucleotide NO. Nucleotide sequence^(a) NO. positionBacterial species: S. aureus 12325′-GGA GCC GCG CGA TTT TAT AAA TGA ATG TTG 1244 53-80 ATA ACC GGC TCC^(a)Underlined nucleotides indicate the molecular beacon's stem.

TABLE 85 Molecular beacon internal hybridization probes forspecific detection of tuf sequences. Originating DNA fragment SEQ IDSEQ ID Nucleotide NO. Nucleotide sequence^(a) NO. positionBacterial species: Chlamydia pneumoniae 20915′-CGC GAC TTG AGA TGG AAC TTA GTG AGC   20 157-183 TTC TTG GTC GCG 20925′-CGC GAC GAA AGA ACT TCC TGA AGG TCG   20 491-516 TGC AGG TCC AGBacterial species: Chlamydia trachomatis 22135′-CGT GCC ATT GAC ATG ATT TCC GAA GAA 1739^(b) 412-441GAC GCT GAA GGC ACG Bacterial species: Enterococcus faecalis 12365′-GCG AGC CGT GGT GAA GTT CGC GTT GGT  883 370-391 GGC TCG CBacterial species: Enterococcus faecium 12355′-GCG AGC CGA AGT TGA AGT TGT TGG TAT   64 412-437 TGC TGG CTC GCBacterial species: Legionella pneumophila 2084^(c)5′-CAC GCG TCA ACA CCC GTA CAA GTC GTC  112 461-486 TTT TGC GCG TGBacterial species: Mycoplasma pneumoniae 2096^(c)5′-CGC GAC CGG TAC CAC GGC CAG TAA TCG 2097^(b) 658-679 TGT CGC GBacterial species: Neisseria gonorrhoeae 21775′-GGC ACG GAC AAA CCA TTC CTG CTG CCT  126 323-357ATC GAA ACG TGT TCC CGT GCC 2178 5′-GGC ACG ACA AAC CAT TCC TGC TGC CTA 126 323-348 TCG AAC GTG CC 2179 5′-GGC AGC TCT ACT TCC GTA CCA CTG ACG 126 692-718 TAA CCG GCT GCC^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)Sequence from databases.^(c)This sequence is from the complementary DNA strand of the sequenceof the originating fragment given in the Sequence Listing.Bacterial species: Pseudomonas aeruginosa 21225′-CCG AGC GAA TGT AGG AGT CCA GGG TCT 153, 880, 2138^(b,c)  280-302^(d)CTG CTC GG Bacterial species: Staphylococcus aureus 21865′-ACG CGC TCA AAG CAG AAG TAT ACG TAT 1728 615-646TAT CAA AAG ACG CGC GTBacterial group: Staphylococcus sp. other than S. aureus 12335′-GCG AGC GTT ACT GGT GTA GAA ATG TTC  878 372-394 CGG CTC GCFungal species: Candida albicans 20735′-CCG AGC AAC ATG ATT GAA CCA TCC ACC  408 404-429 AAC TGG CTC GGFungal species: Candida dubliniensis 20745′-CCG AGC AAC ATG ATT GAA GCT TCC ACC  414 416-441 AAC TGG CTC GGFungal species: Candida glabrata 2110^(b)5′-GCG GGC CCT TAA CGA TTT CAG CGA ATC  417 307-335 TGG ATT CAG CCC GC2111 5′-GCG GGC ATG TTG AAG CCA CCA CCA ACG  417 419-447CTT CCT GGC CCG C Fungal species: Candida krusei 2112^(b)5′-GCG GGC TTG ATG AAG TTT GGG TTT CCT  422 318-347 TGA CAA TTG CCC GC2113 5′-GCG GGC ACA AGG GTT GGA CTA AGG AAA  422 419-447CCA AGG CAG CCC  GC 2114 5′-GCG GGC ATC GAT GCT ATT GAA CCA CCT  422505-533 GTC AGA CCG CCC GC^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)Sequence from databases.^(c)These sequences were aligned to derive the corresponding primer.^(d)The nucleotide positions refer to the P. aeruginosa tuf sequencefragment (SEQ ID NO. 153). Fungal species: Candida lusitaniae 2115^(b)5′-GCG GGC GGT AAG TCC ACC GGT AAG ACC  424 304-330 TTG TTG GCC CGC 21165′-GCG GGC GTA AGT CAC CGG TAA GAC CTT  424 476-502 GTT GGC CCG C 21175′-GCG GGC GAC GCC ATT GAG CCA CCT TCG  424 512-535 AGA GCC CGCFungal species: Candida parapsilosis 2118^(b)5′-GCG GGC TCC TTG ACA ATT TCT TCG TAT  426 301-330 CTG TTC TTG GCC CGCFungal species: Candida tropicalis 21195′-GCG GGC TTA CAA CCC TAA GGC TGT TCC  429 357-384 ATT CGT TGC CCG C2120 5′-GCG GGC AGA AAC CAA GGC TGG TAA GGT  429 459-487TAC CGG AGC CCG C Fungal species: Cryptococcus neoformans 21065′-GCG AGC AGA GCA CGC CCT CCT CGC CGC 623, 1985, 1986^(c)  226-244^(d)TCG C 2107 5′-GCG AGC TCC CCA TCT CTG GTT GGC ACG 623, 1985, 1986^(c) 390-408^(d) CTC GC Bacterial genus: Legionella sp. 20835′-CCG CCG ATG TTC CGT AAA TTA CTT GAI 111-112^(d)  488-519^(e)GAA GGT CGA GCC GGC GG^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)This sequence is from the complementary DNA strand of the sequenceof the originating fragment given in the Sequence Listing.^(c)These sequences were aligned to derive the corresponding primer.^(d)The nucleotide positions refer to the C. neoformans tuf (EF-1) sequencefragment (SEQ ID NO. 623).^(e)The nucleotide positions refer to the L. pneumophila tuf (EF-1) sequencefragment (SEQ ID NO. 112). Fungal genus: Candida sp. 21085′-GCG GGC AAC TTC RTC AAG AAG GTT GGT 414, 417,  52-80^(c)TAC AAC CCG CCC GC 422, 424, 426, 429, 624^(b) 21095′-GCG GGC CCA ATC TCT GGT TGG AAY GGT Same as SEQ  100-125^(c)GAC AAG CCC GC ID NO. 2108 Bacterial group: Pseudomonads 21215′-CGA CCG CIA GCC GCA CAC CAA GTT CCG 153-155,  598-616^(e) GTC G205, 880, 2137^(d), 2138^(d,b)^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)These sequences were aligned to derive the corresponding primer.^(c)The nucleotide positions refer to the C. albicans tuf (EF-1) sequence fragment (SEQ ID NO. 624). ^(d)Sequence from databases.^(e)The nucleotide positions refer to the P. aeruginosa tuf sequence fragment (SEQ ID NO. 153).

TABLE 86 Molecular beacon internal hybridization probes for specificdetection of ddl and mtl gene sequences. Originating DNA fragment SEQSEQ ID Nucleotide ID NO. Nucleotide sequence^(a) NO. positionBacterial species: E. faecium (ddl) 1237 5′-GCG AGC CGC GAA ATC GAA1242^(b) 334-359 GTT GCT GTA TTA GGG CTC GCBacterial species: E. faecalis (mtl) 1238 5′-GCG AGC GGC GTT AAT TTT1243^(b) 631-656 GGC ACC GAA GAA GAG CTC GC ^(a)Underlined nucleotidesindicate the molecular beacon's stem. ^(b)Sequence from databases.

TABLE 87 Internal hybridization probe forspecific detection of S. aureus sequences of unknown coding potential.Originating DNA fragment SEQ SEQ ID ID Nucleotide NO.Nucleotide sequence NO. positionBacterial species: Staphylococcus aureus 12345′-ACT AAA TAA ACG CTC ATT CG 1244 35-54

TABLE 88 Specific and ubiquitous primers for nucleic acidamplification (antimicrobial agents resistance genes sequences).Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Resistance gene: aac(2′)-Ia 13445′-AGC AGC AAC GAT GTT ACG CAG CAG 1348^(a) 163-186 1345^(b)5′-CCC GCC GAG CAT TTC AAC TAT TG 1348^(a) 392-414 13465′-GAT GTT ACG CAG CAG GGC AGT C 1348^(a) 172-193 1347^(b)5′-ACC AAG CAG GTT CGC AGT CAA GTA 1348^(a) 467-490Resistance gene: aac(3′)-Ib 1349 5′-CAG CCG ACC AAT GAG TAT CTT GCC1351^(a) 178-201 1350^(b) 5′-TAA TCA GGG CAG TTG CGA CTC CTA 1351^(a)356-379 Resistance gene: aac(3′)-IIb 13525′-CCA CGC TGA CAG AGC CGC ACC G 1356^(a) 383-404 1353^(b)5′-GGC CAG CTC CCA TCG GAC CCT G 1356^(a) 585-606 13545′-CAC GCT GAC AGA GCC GCA CCG 1356^(a) 384-404 1355^(b)5′-ATG CCG TTG CTG TCG AAA TCC TCG 1356^(a) 606-629Resistance gene: aac(3′)-IVa 1357 5′-GCC CAT CCA TTT GCC TTT GC 1361^(a)295-314 1358^(b) 5′-GCG TAC CAA CTT GCC ATC CTG AAG 1361^(a) 517-5401359 5′-TGC CCC TGC CAC CTC ACT C 1361^(a) 356-374 1360^(b)5′-CGT ACC AAC TTG CCA TCC TGA AGA 1361^(a) 516-539Resistance gene: aac(3′)-VIa 1362 5′-CGC CGC CAT CGC CCA AAG CTG G1366^(a) 285-306 1363^(b) 5′-CGG CAT AAT GGA GCG CGG TGA CTG 1366^(a)551-574 1364 5′-TTT CTC GCC CAC GCA GGA AAA ATC 1366^(a) 502-5251365^(b) 5′-CAT CCT CGA CGA ATA TGC CGC G 1366^(a) 681-702Resistance gene: aac(6′)-Ia 1367 5′-CAA ATA TAC TAA CAG AAG CGT TCA1371^(a) 56-79 1368^(b) 5′-AGG ATC TTG CCA ATA CCT TTA T 1371^(a)269-290 1379 5′-AAA CCT TTG TTT CGG TCT GCT AAT 1371^(a) 153-1761380^(b) 5′-AAG CGA TTC CAA TAA TAC CTT GCT 1371^(a) 320-343Resistance gene: aac(6′)-Ic 1372 5′-GCT TTC GTT GCC TTT GCC GAG GTC1376^(a) 157-180 1373^(b) 5′-CAC CCC TGT TGC TTC GCC CAC TC 1376^(a)304-326 1374 5′-AGA TAT TGG CTT CGC CGC ACC ACA 1376^(a) 104-1271375^(b) 5′-CCC TGT TGC TTC GCC CAC TCC TG 1376^(a) 301-323Resistance gene: ant(3′)-Ia 1377 5′-GCC GTG GGT CGA TGT TTG ATG TTA1381^(a) 100-123 1378^(b) 5′-GCT CGA TGA CGC CAA CTA CCT CTG 1381^(a)221-244 1379 5′-AGC AGC AAC GAT GTT ACG CAG CAG 1381^(a) 127-1501380^(b) 5′-CGC TCG ATG ACG CCA ACT ACC TCT 1381^(a) 222-245Resistance gene: ant(4′)-Ia 1382 5′-TAG ATA TGA TAG GCG GTA AAA AGC1386^(a) 149-172 1383^(b) 5′-CCC AAA TTC GAG TAA GAG GTA TT 1386^(a)386-408 1384 5′-GAT ATG ATA GGC GGT AAA AAG C 1386^(a) 151-172 1385^(b)5′-TCC CAA ATT CGA GTA AGA GGT A 1386^(a) 388-409Resistance gene: aph(3′)-Ia 1387 5′-TTA TGC CTC TTC CGA CCA TCA AGC1391^(a) 233-256 1338^(b) 5′-TAC GCT CGT CAT CAA AAT CAC TCG 1391^(a)488-511 1389 5′-GAA TAA CGG TTT GGT TGA TGC GAG 1391^(a) 468-4911390^(b) 5′-ATG GCA AGA TCC TGG TAT CGG TCT 1391^(a) 669-692Resistance gene: aph(3′)-IIa 1392 5′-TGG GTG GAG AGG CTA TTC GGC TAT1396^(a) 43-66 1393^(b) 5′-CAG TCC CTT CCC GCT TCA GTG AC 1396^(a)250-272 1394 5′-GAC GTT GTC ACT GAA GCG GGA AGG 1396^(a) 244-2671395^(b) 5′-CTT GGT GGT CGA ATG GGC AGG TAG 1396^(a) 386-409Resistance gene: aph(3′)-IIIa 1397 5′-GTG GGA GAA AAT GAA AAC CTA T1401^(a) 103-124 1398^(b) 5′-ATG GAG TGA AAG AGC CTG AT 1401^(a) 355-3741399 5′-ACC TAT GAT GTG GAA CGG GAA AAG 1401^(a) 160-183 1400^(b)5′-CGA TGG AGT GAA AGA GCC TGA TG 1401^(a) 354-376Resistance gene: aph(3′)-VIa 1402 5′-TAT TCA ACA ATT TAT CGG AAA CAG1406^(a) 18-41 1403^(b) 5′-TCA GAG AGC CAA CTC AAC ATT TT 1406^(a)175-197 1404 5′-AAA CAG CGT TTT AGA GCC AAA TAA 1406^(a) 36-59 1405^(b)5′-TTC TCA GAG AGC CAA CTC AAC ATT 1406^(a) 177-200Resistance gene: blaCARB 1407 5′-CCC TGT AAT AGA AAA GCA AGT AGG1411^(a) 351-374 1408^(b) 5′-TTG TCG TAT CCC TCA AAT CAC C 1411^(a)556-577 1409 5′-TGG GAT TAC AAT GGC AAT CAG CG 1411^(a) 205-227 1410^(b)5′-GGG GAA TAG GTC ACA AGA TCT GCT T 1411^(a) 329-353Resistance gene: blaCMY-2 1412 5′-GAG AAA ACG CTC CAG CAG GGC 1416^(a)793-813 1413^(b) 5′-CAT GAG GCT TTC ACT GCG GGG 1416^(a) 975-995 14145′-TAT CGT TAA TCG CAC CAT CAC 1416^(a)  90-110 1415^(b)5′-ATG CAG TAA TGC GGC TTT ATC 1416^(a) 439-459Resistance genes: blaCTX-M-1, blaCTX-M-2 14175′-TGG TTA ACT AYA ATC CSA TTG CGG A 1423^(a) 314-338 1418^(b)5′-ATG CTT TAC CCA GCG TCA GAT T 1423^(a) 583-604Resistance gene: blaCTX-M-1 1419 5′-CGA TGA ATA AGC TGA TTT CTC ACG1423^(a) 410-433 1420^(b) 5′-TGC TTT ACC CAG CGT CAG ATT ACG 1423^(a)580-603 1421 5′-AAT TAG AGC GGC AGT CGG GAG GAA 1423^(a) 116-1391422^(b) 5′-GAA ATC AGC TTA TTC ATC GCC ACG 1423^(a) 405-428Resistance gene: blaCTX-M-2 1424 5′-GTT AAC GGT GAT GGC GAC GCT AC1428^(a) 30-52 1425^(b) 5′-GAA TTA TCG GCG GTG TTA ATC AGC 1428^(a)153-176 1426 5′-CAC GCT CAA TAC CGC CAT TCC A 1428^(a) 510-531 1427^(b)5′-TTA TCG CCC ACT ACC CAT GAT TTC 1428^(a) 687-710Resistance gene: blaIMP 1429 5′-TTT ACG GCT AAA GAT ACT GAA AAG T1433^(a) 205-229 1430^(b) 5′-GTT TAA TAA AAC AAC CAC CGA ATA AT 1433^(a)513-538 1431 5′-TAA TTG ACA CTC CAT TTA CGG CTA A 1433^(a) 191-2151432^(b) 5′-ACC GAA TAA TAT TTT CCT TTC AGG CA 1433^(a) 497-522Resistance gene: blaOXA2 1434 5′-CAC AAT CAA GAC CAA GAT TTG CGA T1438^(a) 319-343 1435^(b) 5′-GAA AGG GCA GCT CGT TAC GAT AGA G 1438^(a)532-556 Resistance gene: blaOXA10 14365′-CAG CAT CAA CAT TTA AGA TCC CCA 1439^(a) 194-217 1437^(b)5′-CTC CAC TTG ATT AAC TGC GGA AAT TC 1439^(a) 479-504Resistance gene: blaPER-1 1440 5′-AGA CCG TTA TCG TAA ACA GGG CTA AG1442^(a) 281-306 1441^(b) 5′-TTT TTT GCT CAA ACT TTT TCA GGA TC 1442^(a)579-604 Resistance gene: blaPER-2 1443 5′-CTT CTG CTC TGC TGA TGC TTG GC1445^(a) 32-54 1444^(b) 5′-GGC GAC CAG GTA TTT TGT AAT ACT GC 1445^(a)304-329 Resistance genes: blaPER-1, blaPER-2 14465′-GGC CTG YGA TTT GTT ATT TGA ACT GGT 1442^(a) 414-440 1447^(b)5′-CGC TST GGT CCT GTG GTG GTT TC 1442^(a) 652-674 14485′-GAT CAG GTG CAR TAT CAA AAC TGG AC 1442^(a) 532-557 1449^(b)5′-AGC WGG TAA CAA YCC TTT TAA CCG CT 1442^(a) 671-696Resistance gene: blaSHV 1883 5′-AGC CGC TTG AGC AAA TTA AAC TA 1900^(a)71-93 1884^(b) 5′-GTA TCC CGC AGA TAA ATC ACC AC 1900^(a) 763-785 18855′-AGC GAA AAA CAC CTT GCC GAC 1900^(a) 313-333 1884^(b)5′-GTA TCC CGC AGA TAA ATC ACC AC 1900^(a) 763-785Resistance gene: blaTEM 1906 5′-CCT TAT TCC CTT TTT TGC GG 1927^(a)27-46 1907^(b) 5′-CAC CTA TCT CAG CGA TCT GTC T 1927^(a) 817-838 19085′-AAC AGC GGT AAG ATC CTT GAG AG 1927^(a) 148-170 1907^(b)5′-CAC CTA TCT CAG CGA TCT GTC T 1927^(a) 817-838 Resistance gene: catI2145 5′-GCA AGA TGT GGC GTG TTA CGG T 2147^(a) 363-384 2146^(b)5′-GGG GCG AAG AAG TTG TCC ATA TT 2147^(a) 484-506Resistance gene: catII 2148 5′-CAG ATT AAA TGC GGA TTC AGC C 2150^(a)67-88 2149^(b) 5′-ATC AGG TAA ATC ATC AGC GGA TA 2150^(a) 151-173Resistance gene: catIII 2151 5′-ATA TTT CAG CAT TAC CTT GGG TT 2153^(a)419-441 2152^(b) 5′-TAC ACA ACT CTT GTA GCC GAT TA 2153^(a) 603-625Resistance gene: catP 2154 5′-CGC CAT TCA GAG TTT AGG AC 2156^(a)178-197 2155^(b) 5′-TTC CAT ACC GTT GCG TAT CAC TT 2156^(a) 339-361Resistance gene: cat 2157 5′-CCA CAG AAA TTG ATA TTA GTG TTT TAT2159^(a)  89-115 2158^(b) 5′-TCG CTA TTG TAA CCA GTT CTA 2159^(a)201-221 2160 5′-TTT TGA ACA CTA TTT TAA CCA GC 2162^(a) 48-70 2161^(b)5′-GAT TTA ACT TAT CCC AAT AAC CT 2162^(a) 231-253 Resistance gene: dfrA1450 5′-ACC ACT GGG AAT ACA CTT GTA ATG GC 1452^(a) 106-131 1451^(b)5′-ATC TAC CTG GTC AAT CAT TGC TTC GT 1452^(a) 296-321Resistance gene: dhfrIa 1457 5′-CAA AGG TGA ACA GCT CCT GTT T 1461^(a)75-96 1458^(b) 5′-TCC GTT ATT TTC TTT AGG TTG GTT AAA 1461^(a) 249-2751459 5′-AAG GTG AAC AGC TCC TGT TT 1461^(a) 77-96 1560^(b)5′-GAT CAC TAC GTT CTC ATT GTC A 1461^(a) 207-228Resistance genes: dhfrIa, dhfrXV 14535′-ATC GAA GAA TGG AGT TAT CGG RAA TG 1461^(a) 27-52 1454^(b)5′-CCT AAA AYT RCT GGG GAT TTC WGG A 1461^(a) 384-408 14555′-CAG GTG GTG GGG AGA TAT ACA AAA 1461^(a) 290-313 1456^(b)5′-TAT GTT AGA SRC GAA GTC TTG GKT AA 1461^(a) 416-441Resistance gene: dhfrIb 1466 5′-AAG CAT TGA CCT ACA ATC AGT GT 1470^(a) 98-120 1467^(b) 5′-AAT ACA ACT ACA TTG TCA TCA TTT GAT 1470^(a) 204-2301468 5′-CGT TAC CCG CTC AGG TTG GAC ATC AA 1470^(a) 183-208 1469^(b)5′-CAT CCC CCT CTG GCT CGA TGT CG 1470^(a) 354-376Resistance gene: dhfrV 1471 5′-GAT AAT GAC AAC GTA ATA GTA TTC CC1475^(a) 208-233 1472^(b) 5′-GCT CAA TAT CAA TCG TCG ATA TA 1475^(a)342-364 1473 5′-TTA AAG CCT TGA CGT ACA ACC AGT GG 1475^(a)  95-1201474^(b) 5′-TGG GCA ATG TTT CTC TGT AAA TCT CC 1475^(a) 300-325Resistance genes: dhfrIb, dhfrV 1462 5′-GCA CTC CCY AAT AGG AAA TAC GC1470^(a) 157-179 1463^(b) 5′-AGT GTT GCT CAA AAA CAA CTT CG 1470^(a)405-427 1464 5′-ACG TTY GAA TCT ATG GGM GCA CT 1470^(a) 139-161 1465^(b)5′-GTC GAT AAG TGG AGC GTA GAG GC 1470^(a) 328-350Resistance gene: dhfrVI 1476 5′-GGC GAG CAG CTC CTA TTC AAA G 1480^(a) 79-100 1477^(b) 5′-TAG GTA AGC TAA TGC CGA TTC AAC A 1480^(a) 237-2611478 5′-GAG AAT GGA GTA ATT GGC TCT GGA TT 1480^(a) 31-56 1479^(b)5′-GCG AAA TAC ACA ACA TCA GGG TCA T 1480^(a) 209-233Resistance gene: dhfrVII 1485 5′-AAA ATG GCG TAA TCG GTA ATG GC 1489^(a)32-54 1486^(b) 5′-CAT TTG AGC TTG AAA TTC CTT TCC TC 1489^(a) 189-2141487 5′-AAT CGA AAA TAT GCA GTA GTG TCG AG 1489^(a) 166-191 1488^(b)5′-AGA CTA TTG TAG ATT TGA CCG CCA 1489^(a) 294-317Resistance genes: dhfrVII, dhfrXVII 14815′-RTT ACA GAT CAT KTA TAT GTC TCT 1489^(a) 268-291 1482^(b)5′-TAA TTT ATA TTA GAC AWA AAA AAC TG 1489^(a) 421-446 14835′-CAR YGT CAG AAA ATG GCG TAA TC 1489^(a) 23-45 1484^(b)5′-TKC AAA GCR WTT TCT ATT GAA GGA AA 1489^(a) 229-254Resistance gene: dhfrVIII 1490 5′-GAC CTA TGA GAG CTT GCC CGT CAA A1494^(a) 144-168 1491^(b) 5′-TCG CCT TCG TAC AGT CGC TTA ACA AA 1494^(a)376-401 1492 5′-CAT TTT AGC TGC CAC CGC CAA TGG TT 1494^(a) 18-431493^(b) 5′-GCG TCG CTG ACG TTG TTC ACG AAG A 1494^(a) 245-269Resistance gene: dhfrIX 1495 5′-TCT CTA AAC ATG ATT GTC GCT GTC 1499^(a) 7-30 1496^(b) 5′-CAG TGA GGC AAA AGT TTT TCT ACC 1499^(a) 133-156 14975′-CGG ACG ACT TCA TGT GGT AGT CAG T 1499^(a) 171-195 1498^(b)5′-TTT GTT TTC AGT AAT GGT CGG GAC CT 1499^(a) 446-471Resistance gene: dhfrXII 1500 5′-ATC GGG TTA TTG GCA ATG GTC CTA1504^(a) 50-73 1501^(b) 5′-GCG GTA GTT AGC TTG GCG TGA GAT T 1504^(a)201-225 1502 5′-GCG GGC GGA GCT GAG ATA TAC A 1504^(a) 304-325 1503^(b)5′-AAC GGA GTG GGT GTA CGG AAT TAC AG 1504^(a) 452-477Resistance gene: dhfrXIII 1505 5′-ATT TTT CGC AGG CTC ACC GAG AGC1507^(a) 106-129 1506^(b) 5′-CGG ATG AGA CAA CCT CGA ATT CTG CTG1507^(a) 413-439 Resistance gene: dhfrXV 15085′-AGA ATG TAT TGG TAT TTC CAT CTA TCG 1512^(a) 215-241 1509^(b)5′-CAA TGT CGA TTG TTG AAA TAT GTA AA 1512^(a) 336-361 15105′-TGG AGT GCC AAA GGG GAA CAA T 1512^(a) 67-88 1511^(b)5′-CAG ACA CAA TCA CAT GAT CCG TTA TCG 1512^(a) 266-292Resistance gene: dhfrXVII 1513 5′-TTC AAG CTC AAA TGA AAA CGT CC1517^(a) 201-223 1514^(b) 5′-GAA ATT CTC AGG CAT TAT AGG GAA T 1517^(a)381-405 1515 5′-GTG GTC AGT AAA AGG TGA GCA AC 1517^(a) 66-88 1516^(b)5′-TCT TTC AAA GCA TTT TCT ATT GAA GG 1517^(a) 232-257Resistance gene: embB 2102 5′-CAC CTT CAC CCT GAC CGA CG 2105^(a)822-841 2103^(b) 5′-CGA ACC AGC GGA AAT AGT TGG AC 2105^(a) 948-970Resistance genes: ereA, ereA2 1528 5′-AAC TTG AGC GAT TTT CGG ATA CCC TG1530^(a)  80-105 1529^(b) 5′-TTG CCG ATG AAA TAA CCG CCG ACT 1530^(a)317-340 Resistance gene: ereB 1531 5′-TCT TTT TGT TAC GAC ATA CGC TTT T1535^(a) 152-176 1532^(b) 5′-AGT GCT TCT TTA TCC GCT GTT CTA 1535^(a)456-479 1533 5′-CAG CGG ATA AAG AAG CAC TAC ACA TT 1535^(a) 461-4861534^(b) 5′-CCT CCT GAA ATA AAG CCC GAC AT 1535^(a) 727-749Resistance gene: gyrA 1340 5′-GAA CAA GGT ATG ACA CCG GAT AAA T 1299^(a)163-188 1341^(b) 5′-GAT AAC TGA AAT CCT GAG CCA TAC G 1299^(a) 274-2991936 5′-TAC CAC CCG CAC GGC 1954^(a) 205-219 1937^(b)5′-CGG AGT CGC CGT CGA TG 1954^(a) 309-325 19425′-GAC TGG AAC AAA GCC TAT AAA AAA TCA 1954^(a) 148-174 1937^(b)5′-CGG AGT CGC CGT CGA TG 1954^(a) 309-325 20405′-TGT GAC CCC AGA CAA ACC C 2054^(a) 33-51 2041^(b)5′-GTT GAG CGG CAG CAC TAT CT 2054^(a) 207-226 Resistance gene: inhA2098 5′-CTG AGT CAC ACC GAC AAA CGT C 2101^(a) 910-931 2099^(b)5′-CCA GGA CTG AAC GGG ATA CGA A 2101^(a) 1074-1095Resistance genes: linA, linA′ 1536 5′-AGA TGT ATT AAC TGG AAA ACA ACA A1540^(a)  99-123 1537^(b) 5′-CTT TGT AAT TAG TTT CTG AAA ACC A 1540^(a)352-376 1538 5′-TTA GAA GAT ATA GGA TAC AAA ATA GAA G 1540^(a) 187-2141539^(b) 5′-GAA TGA AAA AGA AGT TGA GCT T 1540^(a) 404-425Resistance gene: linB 1541 5′-TGA TAA TCT TAT ACG TGG GGA ATT T 1545^(a)246-270 1542^(b) 5′-ATA ATT TTC TAA TTG CCC TGT TTC AT 1545^(a) 359-3841543 5′-GGG CAA TTA GAA AAT TAT TTA TCA GA 1545^(a) 367-392 1544^(b)5′-TTT TAC TCA TGT TTA GCC AAT TAT CA 1545^(a) 579-604Resistance gene: mefA 1546 5′-CAA GAA GGA ATG GCT GTA CTA C 1548^(a)625-646 1547^(b) 5′-TAA TTC CCA AAT AAC CCT AAT AAT AGA 1548^(a) 816-842Resistance gene: mefE 1549 5′-GCT TAT TAT TAG GAA GAT TAG GGG GC1551^(a) 815-840 1550^(b) 5′-TAG CAA GTG ACA TGA TAC TTC CGA 1551^(a)1052-1075 Resistance genes: mefA, mefE 15525′-GGC AAG CAG TAT CAT TAA TCA CTA 1548^(a) 50-73 1553^(b)5′-CAA TGC TAC GGA TAA ACA ATA CTA TC 1548^(a) 318-343 15545′-AGA AAA TTA AGC CTG AAT ATT TAG GAC 1548^(a) 1010-1035 1555^(b)5′-TAG TAA AAA CCA ATG ATT TAC ACC G 1548^(a) 1119-1143Resistance genes: mphA, mphK 1556 5′-ACT GTA CGC ACT TGC AGC CCG ACA T1560^(a) 33-57 1557^(b) 5′-GAA CGG CAG GCG ATT CTT GAG CAT 1560^(a)214-237 1558 5′-GTG GTG GTG CAT GGC GAT CTC T 1560^(a) 583-604 1559^(b)5′-GCC GCA GCG AGG TAC TCT TCG TTA 1560^(a) 855-878Resistance gene: mupA 2142 5′-GCC TTA ATT TCG GAT AGT GC 2144^(a)1831-1850 2143^(b) 5′-GAG AAA GAG CCC AAT TAT CTA ATG T 2144^(a)2002-2026 Resistance gene: parC 1342 5′-GAT GTT ATT GGT CAA TAT CAT CCA1321^(a) 205-229 1343^(b) 5′-AAG AAA CTG TCT CTT TAT TAA TAT CAC GT1321^(a) 396-425 1934 5′-GAA CGC CAG CGC GAA ATT CAA AAA G 1781 67-911935^(b) 5′-AGC TCG GCA TAC TTC GAC AGG 1781 277-297 20445′-ACC GTA AGT CGG CCA AGT CA 2055^(a) 176-195 2045^(b)5′-GTT CTT TCT CCG TAT CGT C 2055^(a) 436-454Resistance gene: ppflo-like 2163 5′-ACC TTC ATC CTA CCG ATG TGG GTT2165^(a) 922-945 2164^(b) 5′-CAA CGA CAC CAG CAC TGC CAT TG 2165^(a)1136-1158 Resistance gene: rpoB 2065 5′-CCA GGA CGT GGA GGC GAT CAC A2072^(a) 1218-1239 2066^(b) 5′-CAC CGA CAG CGA GCC GAT CAG A 2072^(a)1485-1506 Resistance gene: satG 15815′-AAT TGG GGA CTA CAC CTA TTA TGA TG 1585^(a)  93-118 1582^(b)5′-GGC AAA TCA GTC AGT TCA GGA GT 1585^(a) 310-332 15835′-CGA TTG GCA ACA ATA CAC TCC TG 1585^(a) 294-316 1584^(b)5′-TCA CCT ATT TTT ACG CCT GGT AGG AC 1585^(a) 388-413Resistance gene: sulII 1961 5′-GCT CAA GGC AGA TGG CAT TCC C 1965^(a)222-243 1962^(b) 5′-GGA CAA GGC GGT TGC GTT TGA T 1965^(a) 496-517 19635′-CAT TCC CGT CTC GCT CGA CAG T 1965^(a) 237-258 1964^(b)5′-ATC TGC CTG CCC GTC TTG C 1965^(a) 393-411 Resistance gene: tetB 19665′-CAT GCC AGT CTT GCC AAC G 1970^(a) 66-84 1967^(b)5′-CAG CAA TAA GTA ATC CAG CGA TG 1970^(a) 242-264 19685′-GGA GAG ATT TCA CCG CAT AG 1970^(a) 457-476 1969^(b)5′-AGC CAA CCA TCA TGC TAT TCC A 1970^(a) 721-742 Resistance gene: tetM1586 5′-ATT CCC ACA ATC TTT TTT ATC AAT AA 1590^(a) 361-386 1587^(b)5′-CAT TGT TCA GAT TCG GTA AAG TTC 1590^(a) 501-524 15885′-GTT TTT GAA GTT AAA TAG TGT TCT T 1590^(a) 957-981 1589^(b)5′-CTT CCA TTT GTA CTT TCC CTA 1590^(a) 1172-1192 Resistance gene: vatB1609 5′-GCC CTG ATC CAA ATA GCA TAT A 1613^(a) 11-32 1610^(b)5′-CCT GGC ATA ACA GTA ACA TTC TG 1613^(a) 379-401 16115′-TGG GAA AAA GCA ACT CCA TCT C 1613^(a) 301-322 1612^(b)5′-ACA ACT GAA TTC GCA GCA ACA AT 1613^(a) 424-446 Resistance gene: vatC1614 5′-CCA ATC CAG AAG AAA TAT ACC C 1618^(a) 26-47 1615^(b)5′-ATT AGT TTA TCC CCA ATC AAT TCA 1618^(a) 177-200 16165′-ATA ATG AAT GGG GCT AAT CAT CGT AT 1618^(a) 241-266 1617^(b)5′-GCC AAC AAC TGA ATA AGG ATC AAC 1618^(a) 463-486 Resistance gene: vga1619 5′-AAG GCA AAA TAA AAG GAG CAA AGC 1623^(a) 641-664 1620^(b)5′-TGT ACC CGA GAC ATC TTC ACC AC 1623^(a) 821-843 16215′-AAT TGA AGG ACG GGT ATT GTG GAA AG 1623^(a) 843-868 1622^(b)5′-CGA TTT TGA CAG ATG GCG ATA ATG AA 1623^(a)  975-1000Resistance gene: vgaB 1624 5′-TTC TTT AAT GCT CGT AGA TGA ACC TA1628^(a) 354-379 1625^(b) 5′-TTT TCG TAT TCT TCT TGT TGC TTT C 1628^(a)578-602 1626 5′-AGG AAT GAT TAA GCC CCC TTC AAA AA 1628^(a) 663-6881627^(b) 5′-TTA CAT TGC GAC CAT GAA ATT GCT CT 1628^(a) 849-874Resistance genes: vgb, vgh 1629 5′-AAG GGG AAA GTT TGG ATT ACA CAA CA1633^(a) 73-98 1630^(b) 5′-GAA CCA CAG GGC ATT ATC AGA ACC 1633^(a)445-468 1631 5′-CGA CGA TGC TTT ATG GTT TGT 1633^(a) 576-596 1632^(b)5′-GTT AAT TTG CCT ATC TTG TCA CAC TC 1633^(a) 850-875Resistance gene: vgbB 1634 5′-TTA ACT TGT CTA TTC CCG ATT CAG G 1882^(a)23-47 1635^(b) 5′-GCT GTG GCA ATG GAT ATT CTG TA 1882^(a) 267-289 16365′-TTC CTA CCC CTG ATG CTA AAG TGA 1882^(a) 155-178 1637^(b)5′-CAA AGT GCG TTA TCC GAA CCT AA 1882^(a) 442-464 Sequencing primersResistance gene: gyrA 1290 5′-GAY TAY GCI ATG ISI GTI ATH GT 1299^(a)70-83 1292^(b) 5′-ARI SCY TCI ARI ATR TGI GC 1299^(a) 1132-1152 12915′-GCI YTI CCI GAY GTI MGI GAY GG 1299^(a) 100-123 1292^(b)5′-ARI SCY TCI ARI ATR TGI GC 1299^(a) 1132-1152 12935′-ATG GCT GAA TTA CCT CAA TC 1299^(a)  1-21 1294^(b)5′-ATG ATT GTT GTA TAT CTT CTT CAA C 1299^(a) 2626-2651 1295^(b)5′-CAG AAA GTT TGA AGC GTT GT 1299^(a) 1255-1275 12965′-AAC GAT TCG TGA GTC AGA TA 1299^(a) 1188-1208 12975′-CGG TCA ACA TTG AGG AAG AGC T 1300^(a) 29-51 1298^(b)5′-ACG AAA TCG ACC GTC TCT TTT TC 1300^(a) 415-437 Resistance gene: gyrB1301 5′-GTI MGI AWI MGI CCI GSI ATG TA 1307^(a)  82-105 1302^(b)5′-TAI ADI GGI GGI KKI GCI ATR TA 1307^(a) 1600-1623 13035′-GGI GAI GAI DYI MGI GAR GG 1307^(a) 955-975 1304^(b)5′-CIA RYT TIK YIT TIG TYT G 1307^(a) 1024-1043 13055′-ATG GTG ACT GCA TTG TCA GAT G 1307^(a)  1-23 1306^(b)5′-GTC TAC GGT TTT CTA CAA CGT C 1307^(a) 1858-1888Resistance gene: parC 1308 5′-ATG TAY GTI ATI ATG GAY MGI GC 1320^(a)67-90 1309^(b) 5′-ATI ATY TTR TTI CCY TTI CCY TT 1320^(a) 1993-2016 13105′-ATI ATI TSI ATI ACY TCR TC 1320^(a) 1112-1132 1311^(b)5′-GAR ATG AAR ATI MGI GGI GAR CA 1320^(a) 1288-1311 13125′-AAR TAY ATI ATI CAR GAR MGI GC 1321^(a) 67-90 1313^(b)5′-AMI AYI CKR TGI GGI TTI TTY TT 1321^(a) 2212-2235 13145′-TAI GAI TTY ACI GAI SMI CAR GC 1321^(a) 1228-1251 1315^(b)5′-ACI ATI GCI TCI GCY TGI KSY TC 1321^(a) 1240-1263 13165′-GTG AGT GAA ATA ATT CAA GAT T 1321^(a)  1-23 1317^(b)5′-CAC CAA AAT CAT CTG TAT CTA C 1321^(a) 2356-2378 13185′-ACC TAY TCS ATG TAC GTR ATC ATG GA 1320^(a) 58-84 1319^(b)5′-AGR TCG TCI ACC ATC GGY AGY TT 1320^(a) 832-855 Resistance gene: parE1322 5′-RTI GAI AAY ISI GTI GAY GAR G 1328^(a) 133-155 1325^(b)5′-RTT CAT YTC ICC IAR ICC YTT 1328^(a) 1732-1752 13235′-ACI AWR SAI GGI GGI ACI CAY G 1328^(a) 829-850 1324^(b)5′-CCI CCI GCI SWR TCI CCY TC 1328^(a) 1280-1302 13265′-TGA TTC AAT ACA GGT TTT AGA G 1328^(a) 27-49 1327^(b)5′-CTA GAT TTC CTC CTC ATC AAA T 1328^(a) 1971-1993 ^(a)Sequence fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.

TABLE 89 Internal hybridization probes for specific detectionof antimicrobial agents resistance genes sequences. OriginatingDNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO.position Resistance gene: aph3′VIa 2252 5′-CCA CAT ACA GTG TCT CTC1406^(a) 149-166 Resistance gene: blaSHV 1886 5′-GAC GCC CGC GCC ACC ACT1900^(a) 484-501 1887 5′-GAC GCC CGC GAC ACC ACT A 1899^(a) 514-532 18885′-GAC GCC CGC AAC ACC ACT A 1901^(a) 514-532 18895′-GTT CGC AAC TGC AGC TGC TG 1899^(a) 593-612 18905′-TTC GCA ACG GCA GCT GCT G 1899^(a) 594-612 18915′-CCG GAG CTG CCG AIC GGG 1902^(a) 692-709 18925′-CGG AGC TGC CAA RCG GGG 1903^(a) 693-710 18935′-GGA GCT GGC GAR CGG GGT 1899^(a) 694-711 18945′-GAC CGG AGC TAG CGA RCG 1904^(a) 690-707 18955′-CGG AGC TAG CAA RCG GGG T 1905^(a) 693-711 18965′-GAA ACG GAA CTG AAT GAG GCG 1899^(a) 484-504 18975′-CAT TAC CAT GGG CGA TAA CAG 1899^(a) 366-386 18985′-CCA TTA CCA TGA GCG ATA ACA G 1899^(a) 365-386Resistance gene: blaTEM 1909 5′-ATG ACT TGG TTA AGT ACT CAC C 1928^(a)293-314 1910 5′-ATG ACT TGG TTG AGT ACT CAC C 1927^(a) 293-314 19115′-CCA TAA CCA TGG GTG ATA ACA C 1928^(a) 371-392 19125′-CCA TAA CCA TGA GTG ATA ACA C 1927^(a) 371-392 19135′-CGC CTT GAT CAT TGG GAA CC 1928^(a) 475-494 19145′-CGC CTT GAT CGT TGG GAA CC 1927^(a) 475-494 19155′-CGC CTT GAT AGT TGG GAA CC 1929^(a) 475-494 19165′-CGT GGG TCT TGC GGT ATC AT 1927^(a) 712-731 19175′-CGT GGG TCT GGC GGT ATC AT 1930^(a) 712-731 19185′-GTG GGT CTC ACG GTA TCA TTG 1927^(a) 713-733 19195′-CGT GGG TCT CTC GGT ATC ATT 1931^(a) 712-732 19205′-CGT GGI TCT CGC GGT ATC AT 1927^(a) 712-731 19215′-CGT GGG TCT AGC GGT ATC ATT 1932^(a) 713-733 19225′-GTT TTC CAA TGA TTA GCA CTT TTA 1927^(a) 188-211 19235′-GTT TTC CAA TGA TAA GCA CTT TTA 1927^(a) 188-211 19245′-GTT TTC CAA TGC TGA GCA CTT TT 1932^(a) 188-210 19255′-CGT TTT CCA ATG ATG AGC ACT TT 1927^(a) 187-209 19265′-GTT TTC CAA TGG TGA GCA CTT TT 1933^(a) 188-210 20065′-TGG AGC CGG TGA GCG TGG 1927^(a) 699-716 20075′-TGG AGC CAG TGA GCG TGG 2010^(a) 699-716 20085′-TCT GGA GCC GAT GAG CGT G 1929^(a) 697-715 20095′-CTG GAG CCA GTA AGC GTG G 2011^(a) 698-716 21415′-CAC CAG TCA CAG AAA AGC 1927^(a) 311-328 Resistance gene: dhfrIa 22535′-CAT TAC CCA ACC GAA AGT A 1461^(a) 158-176 Resistance gene: embB 21045′-CTG GGC ATG GCI CGA GTC 2105^(a) 910-927 Resistance gene: gyrA 13335′-TCA TGG TGA CTT ATC TAT TTA TG 1299^(a) 240-263 13345′-CAT CTA TTT ATA AAG CAA TGG TA 1299^(a) 251-274 13355′-CTA TTT ATG GAG CAA TGG T 1299^(a) 254-273 19405′-GTA TCG TTG GTG ACG TAA T 1299^(a) 206-224 19435′-GCT GGT GGA CGG CCA G 1954^(a) 279-294 1945 5′-CGG CGA CTA CGC GGT AT1954^(a) 216-232 1946 5′-CGG CGA CTT CGC GGT AT 1954^(a) 216-232 19475′-CGG TAT ACG GCA CCA TCG T 1954^(a) 227-245 19485′-GCG GTA TAC AAC ACC ATC G 1954^(a) 226-244 19495′-CGG TAT ACG CCA CCA TCG T 1954^(a) 227-245 20425′-CAC GGG GAT TTC TCT ATT TA 2054^(a) 103-122 20435′-CAC GGG GAT TAC TCT ATT TA 2054^(a) 103-122 Resistance gene: inhA2100 5′-GCG AGA CGA TAG GTT GTC 2101^(a) 1017-1034 Resistance gene: parC1336 5′-TGG AGA CTA CTC AGT GT 1321^(a) 232-249 13375′-TGG AGA CTT CTC AGT GT 1321^(a) 232-249 1338 5′-GTG TAC GGA GCA ATG1321^(a) 245-260 1339 5′-CCA GCG GAA ATG CGT 1321^(a) 342-357 19415′-GCA ATG GTC CGT TTA AGT 1321^(a) 253-270 19445′-TTT CGC CGC CAT GCG TTA C 1781 247-265 1950 5′-GGC GAC ATC GCC TGC1781 137-151 1951 5′-GGC GAC AGA GCC TGC TA 1781 137-153 19525′-CCT GCT ATG GAG CGA TGG T 1781 147-165 19535′-CGC CTG CTA TAA AGC GAT GGT 1781 145-165 20465′-ACG GGG ATT TTT CTA TCT AT 2055^(a) 227-246 Resistance gene: rpoB2067 5′-AGC TGA GCC AAT TCA TGG 2072^(a) 1304-1321 20685′-ATT CAT GGA CCA GAA CAA C 2072^(a) 1314-1332 20695′-CGC TGT CGG GGT TGA CCC 2072^(a) 1334-1351 20705′-GTT GAC CCA CAA GCG CCG 2072^(a) 1344-1361 20715′-CGA CTG TCG GCG CTG GGG 2072^(a) 1360-1377 Resistance gene: tetM 22545′-ACC TGA ACA GAG AGA AAT G 1590^(a) 1062-1080 ^(a)Sequence fromdatabases.

TABLE 90 Molecular beacon internal hybridization probes forspecific detection of atpD sequences. Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence^(a) NO. positionBacterial species: Bacteroides fragilis 21365′-CCA ACG CGT CCT CAA TCA TTT CTA ACT TCT 929 353-382ATG GCC GGC GTT GG Bacterial species: Bordetella pertussis 21825′-GCG CGC CAA CGA CTT CTA CCA CGA AAT GGA 1672 576-605 AGA GTC GCG CGCBacterial group: Campylobacter jejuni and C. coli 21335′-CCA CGC ACA WAA ACT TGT TTT AGA AGT 1576,  44-73^(d)AGC AGC WCA GCG TGG 1600, 1849, 1863, 2139^(b,c)Fungal species: Candida glabrata 20785′-CCG AGC CTT GGT CTT CGG CCA AAT GAA CGC 463 442-463 TCG GFungal species: Candida krusei 20755′-CCG AGC CAG GTT CTG AAG TCT CTG CAT TAT 468 720-748 TAG GTG CTC GGFungal species: Candida lusitaniae 20805′-CCG AGC CGA AGA GGG CCA AGA TGT CGC TCG G 470 520-538Fungal species: Candida parapsilosis 20795′-CCG AGC GTT CAG TTA CTT CAG TCC AAG CCG 472 837-860 GCT CGGFungal species: Candida tropicalis 20775′-CCG AGC AAC CGA TCC AGC TCC AGC TAC GCT 475 877-897 CGGBacterial species: Klebsiella pneumoniae 22815′-CCC CCA GCT GGG CGG CGG TAT CGA TGG GGG 317 40-59^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)Sequence from databases.^(c)These sequences were aligned to derive the corresponding primer.^(d)The nucleotide positions refer to the C. jejuni atpD sequence fragment(SEQ ID NO. 1576). Fungal genus: Candida sp. 20765′-CCG AGC YGA YAA CAT TTT CAG ATT CAC CCA 460-478,  697-723^(c)RGC GCT CGG 663^(b)^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)These sequences were aligned to derive the corresponding primer.^(c)The nucleotide positions refer to the C. albicans atpD sequence fragment(SEQ ID NO. 460).

TABLE 91 Internal hybridization probes forspecific detection of atpD sequences. Originating DNA fragment SEQSEQ ID Nucleotide ID NO. Nucleotide sequence NO. positionBacterial species: Acinetobacter baumannii 21695′-CCC GTT TGC GAA AGG TGG 243 304-321Bacterial species: Klebsiella pneumoniae 2167 5′-CAG CAG CTG GGC GGC GGT317 36-53

TABLE 92 Internal hybridization probes forspecific detection of ddl and mtl sequences. Originating DNA fragmentSEQ SEQ ID Nucleotide ID NO. Nucleotide sequence NO. positionBacterial species: Enterococcus faecium (dd1) 22865′-AGT TGC TGT ATT AGG 2288^(a) 784-803 AAA TG 22875′-TCG AAG TTG CTG TAT 2288^(a) 780-799 TAG GABacterial species: Enterococcus faecalis mt1) 22895′-CAC CGA AGA AGA TGA 1243^(a) 264-283 AAA AA 22905′-TGG CAC CGA AGA AGA TGA 1243^(a) 261-278 2291 5′-ATT TTG GCA CCG AAG1243^(a) 257-275 AAG A ^(a)Sequence from databases.

What is claimed is:
 1. A method for specifically detecting the presenceor absence in a sample of a tuf nucleic acid sequence belonging to acoagulase negative Staphylococcus comprising: i) contacting the samplewith an oligonucleotide probe or primer that hybridizes to a tuf nucleicacid sequence of each of SEQ ID NOs: 181, 182, 184-188, and 190-202 atnucleotides corresponding to nucleotides 313-679 of SEQ ID NO: 179, orcomplements thereof, but does not hybridize to a tuf nucleic acidsequence of any of SEQ ID NOs: 176-180 under stringent hybridizationconditions of 55° C., 1.5 M NaCl and 10 mM EDTA; ii) allowing saidoligonucleotide to hybridize with said sample under conditions such thatsaid oligonucleotide hybridizes to a tuf nucleic acid sequence of any ofSEQ ID NOs: 181, 182, 184-188, and 190-202 at nucleotides correspondingto nucleotides 313-679 of SEQ ID NO: 179, or complements thereof, ifpresent, but does not hybridize to a tuf nucleic acid sequence of any ofSEQ ID NOs: 176-180 if present; and iii) testing for hybridization ofsaid oligonucleotide to a tuf nucleic acid sequence of SEQ ID NOs: 181,182, 184-188, and 190-202 or complements thereof in said sample.
 2. Themethod of claim 1, wherein the nucleic acid sequence of saidoligonucleotide comprises SEQ ID NO: 1175 or 1176 or a complementthereof.
 3. The method of claim 1, further comprising amplifying saidtuf nucleic acid prior to hybridization of said oligonucleotide to saidtuf nucleic acid.
 4. The method of claim 3, wherein said tuf nucleicacid is amplified using a first primer comprising the nucleic acidsequence of SEQ ID NO: 553 and a second primer comprising a nucleic acidsequence of SEQ ID NO: 575 or
 707. 5. The method of claim 3, whereinsaid oligonucleotide is a probe comprising the nucleic acid sequence ofSEQ ID NO: 1175 or
 1176. 6. The method of claim 5, wherein saidoligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 1233.7. The method of claim 4, wherein said oligonucleotide is a probecomprising the nucleic acid sequence of SEQ ID NO: 1175 or
 1176. 8. Themethod of claim 7, wherein said oligonucleotide comprises the nucleicacid sequence of SEQ ID NO:
 1233. 9. The method of claim 1, wherein thenucleic acid sequence of said oligonucleotide consists of SEQ ID NO:1175 or 1176 or a complement thereof.